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• W2097780147 abstract "Article23 August 2007free access The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation Ioanna Bethani Ioanna Bethani Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany International Max Planck Research School Neurosciences, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Thorsten Lang Thorsten Lang Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Ulf Geumann Ulf Geumann Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Jochen J Sieber Jochen J Sieber Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Reinhard Jahn Corresponding Author Reinhard Jahn Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Silvio O Rizzoli Silvio O Rizzoli Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Ioanna Bethani Ioanna Bethani Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany International Max Planck Research School Neurosciences, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Thorsten Lang Thorsten Lang Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Ulf Geumann Ulf Geumann Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Jochen J Sieber Jochen J Sieber Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Reinhard Jahn Corresponding Author Reinhard Jahn Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Silvio O Rizzoli Silvio O Rizzoli Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Author Information Ioanna Bethani1,2, Thorsten Lang1, Ulf Geumann1, Jochen J Sieber1, Reinhard Jahn 1 and Silvio O Rizzoli1 1Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany 2International Max Planck Research School Neurosciences, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany *Corresponding author. Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany. Tel.: +49 551 201 1635; Fax: +49 551 201 1639; E-mail: [email protected] The EMBO Journal (2007)26:3981-3992https://doi.org/10.1038/sj.emboj.7601820 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in ‘trans’ interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites. Introduction Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are widely regarded as major players in the fusion of intracellular membranes. They constitute a superfamily of membrane-associated proteins characterized by the presence of at least one SNARE motif, which is typically found in the immediate vicinity of the transmembrane domain of the protein. SNARE motifs are classified into four subfamilies, termed Qa-, Qb-, Qc- and R-SNAREs. To catalyze fusion, the SNAREs from two apposing membranes interact in a ‘trans’-configuration. Their SNARE motifs assemble into bundles of four α-helices, and each bundle invariantly contains one SNARE motif of each subfamily. Bundle formation is initiated at the N-terminal end of the SNARE motif, and continues toward the C-terminal end (toward the membrane), thus bringing the two membranes together. After fusion, all members of the complex are found in the same membrane (‘cis’-configuration). Disassembly of the complex requires the activity of the AAA-ATPase NSF that separates the monomers and allows them to engage in subsequent fusion steps (see reviews in Jahn et al, 2003; Hong, 2005; Jahn and Scheller, 2006). Different SNAREs seem to participate in fusion events involving different processes and organelles. It is generally accepted that each fusion step of the secretory pathway requires a specific set of SNAREs, although some SNAREs are known to operate in different fusion steps and others are able to substitute for each other. However, it is unclear by which mechanisms it is ensured that only ‘cognate’ SNAREs interact with each other for fusion. SNAREs associate in vitro with little discrimination between cognate and non-cognate sets, as long as a member of each subclass is present (see review by Jahn and Scheller, 2006). Similar promiscuity was observed in in vitro fusion assays when mammalian SNAREs were reconstituted in artificial vesicles (Brandhorst et al, 2006). The question how cognate SNAREs are selected for fusion is particularly relevant for recycling compartments such as the early endosomes, which constitute a major hub in the endocytotic limb of the secretory pathway. Early endosomes communicate with the trans-Golgi network, with recycling endosomes and late endosomes (Maxfield and McGraw, 2004). Consequently, they contain not only SNAREs mediating endosome fusion, but also additional sets of SNAREs that are ‘passengers’ en route to their resident compartment, including those mediating exocytosis and fusion of late endosomes (Brandhorst et al, 2006). Despite being present in the same membrane, the different sets of SNAREs are functionally well differentiated in the respective fusion steps. For instance, fusion of early endosomes can be competed for by the soluble parts of the cognate, but not of the neuronal SNAREs (Brandhorst et al, 2006). Conversely, cleavage by botulinum or tetanus neurotoxin of any of the neuronal SNAREs blocks synaptic vesicle fusion (see review by Humeau et al, 2000), although the endosomal SNAREs are present on these vesicles (Antonin et al, 2000; Rizzoli et al, 2006). In the present study, we have used a combination of approaches in order to shed light on the mechanisms that govern cognate and non-cognate SNARE interactions in native and fusion-competent membranes. Using early endosomal and neuronal SNAREs as example, we find that within the endosomal membrane, these SNAREs not only are concentrated in overlapping microdomains, but are able to promiscuously form cognate and non-cognate cis-complexes. The proportions of the cognate and non-cognate complexes are dependent on the relative amounts of the individual SNAREs and, as revealed by simulations, are primarily being determined by mass action. When probing for trans-SNARE interactions by addition of soluble SNARE molecules onto native membranes, a certain level of specificity was uncovered, as shown by a preferential binding of cognate SNAREs to their SNARE partners in native membranes. Finally, we observed that at the contact site of fusing endosomes there is lateral segregation of SNAREs, with the exocytotic SNARE synaptobrevin being depleted. We conclude that there is little specificity in complex formation for SNAREs residing in the same membrane, and thus many published SNARE complexes that were previously identified by coprecipitation may not represent functional sets of SNAREs. Trans-specificity is mediated (i) by a preference (but not absolute specificity) for the cognate SNARE in the trans-configuration, probably involving assistance by proof-reading proteins such as SM-proteins, and (ii) by lateral segregation of SNAREs at the contact site of fusing organelles. Results Cognate and non-cognate SNAREs accumulate in common microdomains on the endosomal membrane Early endosomes of neuroendocrine PC12 (pheochromocytoma) cells contain both the SNAREs mediating homotypic fusion (including VAMP4 (R), syntaxin 13 (Qa), syntaxin 6 (Qb) and vti1a (Qc)) and the SNAREs mediating regulated exocytosis of secretory vesicles (including synaptobrevin/VAMP2 (R), syntaxin 1 (Qa) and SNAP-25 (Qbc)). Selective cleavage of the three exocytotic SNAREs blocks exocytosis (Humeau et al, 2000), showing that the early endosomal SNAREs cannot substitute for their exocytotic counterparts in this reaction. Conversely, cleavage of SNAP-25 with BoNT/E, or of syntaxin 1 with BoNT/C1 does not inhibit fusion of early endosomes (Brandhorst et al, 2006; Rizzoli et al, 2006). Cleaving the remaining exocytotic SNARE (synaptobrevin) with tetanus neurotoxin also had no effect on fusion (Supplementary Figure 1). Thus, there is no functional crosstalk between exocytotic and endosomal SNAREs. Although both sets of SNAREs share the same membrane, it is conceivable that their interaction is prevented by lateral segregation in separate microdomains. Such segregation has recently been observed for plasma membrane-resident syntaxins that participate in separate SNARE complexes (Low et al, 2006; Sieber et al, 2006). Since early endosomes are too small to allow discrimination of microdomains by conventional light microscopy, we transfected PC12 cells with a constitutively active mutant of the endosomal GTPase Rab5 (Rab5-Q79L) that was tagged with GFP. Very large endosomes are generated due to increased homotypic fusion activity (Stenmark et al, 1994), which are imaged easily (Figure 1A). The cells were then immunostained for pairs of both cognate and non-cognate SNAREs (Figure 1B–D). An uneven, punctate staining pattern was obtained that was similar between the two SNAREs and contrasted with the more even distribution of GFP-Rab5-Q79L (Figure 1E). To score for colocalization between different SNAREs, we performed confocal sections around the equator of the endosomes, followed by cross-correlation analysis along the endosomal membrane (Figure 1F; see Materials and methods). All SNARE pairs investigated showed a high degree of cross-correlation (although somewhat less for the SNAP-25/vti1a pair), documenting that within the resolution limits of the confocal microscope, there is no appreciable difference in localization between cognate and non-cognate SNARE pairs (Figure 1G). Figure 1.The cognate and non-cognate SNAREs largely colocalize in microdomains on the endosomal membrane. (A) PC12 cells were transfected with a plasmid expressing GFP-Rab5-Q79L. Forty-eight hours post-transfection, cells were fixed and imaged by use of a Zeiss Axiovert 200M fluorescence microscope. The GFP-bound Rab5 variant was observed on vesicular structures of 1–5 μm, which correspond to enlarged early endosomes. Scale bar, 5 μm. (B–D) PC12 cells expressing the GFP-Rab5-Q79L (green) were stained for syntaxin 6 (red channel) and vti1a (blue channel), and imaged by confocal fluorescence microscopy. Images show a typical endosome. Note the SNARE domains on the endosomal membrane. Scale bar, 1 μm. (E) The intensity images in panels B–D are plotted as surfaces, in pseudocolor. Note that the SNAREs are found in domains, which largely correlate. (F) Intensity profiles of syntaxin 6 (red), vti1a (blue) and GFP-Rab5-Q79L (green) signals along the endosomal membrane of the endosome shown in panels B–E. (G) Correlation between the staining of different pairs of SNAREs on the endosomal membrane. Control, costaining of single SNAREs with Cy3- and Cy5-conjugated secondary antibodies. Values are means±s.e. of three independent experiments with 10–15 analyzed endosomes. The striped bars correspond to the negative control (see Materials and methods). Download figure Download PowerPoint Cognate and non-cognate SNAREs form complexes in the membrane of early endosomes Considering that the neuronal SNAREs play no role in endosome fusion, although they are concentrated together with their endosomal counterparts in overlapping microdomains, the question arises whether these SNAREs can form promiscuous complexes (as they do in vitro) or whether native membranes possess mechanisms preventing such non-cognate interactions. To answer this question, we purified early endosomes by discontinuous sucrose gradient centrifugation, resulting in high enrichment of endosomal markers such as Rab5 (Figure 2A), followed by solubilization in Triton X-100. Synaptobrevin was then immunoprecipitated, and the precipitate was analyzed for the presence of both neuronal and early endosomal SNAREs. The results are shown in Figure 2B–C. Both endosomal (syntaxin 6, syntaxin 13, vti1a) and neuronal (SNAP-25 and syntaxin 1) Q-SNAREs were identified in the precipitates, and no strong preference for the cognate SNAREs was observable. The R-SNARE VAMP4 does not precipitate with synaptobrevin, which is to be expected if the cosedimenting SNAREs represent bona-fide four helix bundles with a QabcR composition (see also below). Figure 2.Interactions between non-cognate SNAREs in early endosomes. (A) Enrichment of an early endosomal-specific marker (Rab5) in the fraction isolated from the gradient. Equal amounts of the endosomal fraction (lane1) and post nuclear supernatant (PNS) (lane 2) were analyzed by SDS–PAGE and blotted with anti-Rab5 antibody. For comparison, actin and the NMDA receptor (as a cellular membrane marker) are also shown; the enrichment of endosomes (Rab5 enrichment) was approximately 9.6-fold, on average (±3.9, four experiments). (B) Synaptobrevin interacts with the early endosomal SNAREs. PNS from PC12 cells was centrifuged in a sucrose density gradient, a band highly enriched in early endosomes was isolated, and after solubilization with Triton X-100 immunoprecipitation was performed with a monoclonal antibody against synaptobrevin (left lane); controls are shown in the second lane (no antibody added). The corresponding supernatants (Sup) are shown in the respective position (lanes 3–4). The interacting SNAREs were identified by immunoblot analysis. A total of 10% of the total sample is loaded in every lane. Typical blots of three independent experiments are shown. (C) Quantification by densitometry of coprecipitation, from the blots presented in panel B; the band intensities were normalized to starting material (see Materials and methods). The ‘no antibody’ value corresponds to the mean value of all negative controls presented. (D) Other SNARE interactions in the early endosomal fraction. Immunoprecipitations were performed with antibodies against the SNAREs indicated at the top of the figure. All immunoprecipitates were analyzed by immunoblotting for the SNAREs indicated. The ‘no ab’ value corresponds to the negative control, in which no antibody was added for immunoprecipitation. A total of 10% of the total sample is loaded in every lane. Boxed bands indicate immunoprecipitation of the blotted protein with its own antibody. Empty arrowheads indicate cognate interactions; full arrowheads indicate non-cognate interactions. Note that SNAP-25 was omitted from the analysis because none of the available antibodies detects SNAP-25 in assembled SNARE complexes. Immunoprecipitation of syntaxin 13 by vti1a and syntaxin 1 is presented from PNS material; identical results were obtained with the endosomal fraction (data not shown). See Supplementary Table 1 for the immunoprecipitation efficiencies of all antibodies. Download figure Download PowerPoint Next, we immunoprecipitated VAMP4, syntaxin 13, syntaxin 6, vti1a and syntaxin 1 and analyzed the precipitates for the presence of the other SNAREs (SNAP-25 was omitted because none of a battery of antibodies were capable of precipitating SNAP-25 in complex with other SNAREs). Representative blots are shown in Figure 2D. As for synaptobrevin, no preference for cognate SNAREs was detectable. For instance, traces of SNAP-25 were detected in the precipitates of VAMP4, syntaxin 13 and syntaxin 6 (non-cognate). Conversely, interactions were seen between syntaxin 6 and VAMP4, vti1a and syntaxin 6, and syntaxin 1 and SNAP-25, that all are considered to represent cognate SNARE complexes. Do the co-precipitating SNAREs represent genuine SNARE complexes or nonspecific associations mediated, for instance, by the transmembrane domains? SNARE complexes are disassembled by the AAA-ATPase NSF that is abundantly present in the cytosol. Therefore, we incubated post-nuclear supernatants (PNSs), spiked with rat brain cytosol, to provide additional NSF, in the presence and absence of ATP, before solubilization and immunoprecipitation. Figure 3 shows the results for syntaxin 13, an early endosomal SNARE, for which relatively strong non-cognate interactions were observed (see Supplementary Figure 2 for corresponding results obtained for syntaxin 6). Essentially, no coprecipitating bands are detected in the presence of ATP, whereas in the absence of ATP, interactions between syntaxin 13 and synaptobrevin are evident (Figure 3A, arrowhead). We quantified the immunoprecipitation results obtained in three independent experiments (Figure 3B). Interestingly, syntaxin 13 is associated more strongly with synaptobrevin than with its cognate SNARE partners. ATP preincubation eliminates all of the interactions, both cognate and non-cognate. To confirm that the ATP effects are due to the action of NSF, reactions were carried out in the presence of ATP and the NSF inhibitor N-ethylmaleimide (NEM). Again, the interaction between synaptobrevin and syntaxin 13 was evident (Figure 3C), although the degree of coprecipitation was lower than in the case of ATP depletion (which has also been observed in other systems; Carr et al, 1999). Very similar results were obtained when a rat brain fraction enriched in synaptic vesicles was used (Supplementary Figure 3), which is known to contain full complements of early endosomal and neuronal SNAREs (Rizzoli et al, 2006). Finally, to ensure that the non-cognate interactions are not only observable in isolated organelles, immunoprecipitations were carried out with intact PC12 cells as starting material, which were preincubated in the presence or absence of NEM as an NSF inhibitor. Again, non-cognate complexes were precipitated when NSF was inhibited (Supplementary Figure 3). Figure 3.The non-cognate SNARE interactions represent genuine SNARE complexes in endosomal membranes. (A) PNSs were incubated with rat brain cytosol in presence or absence of ATP, and immunoprecipitations were performed after detergent extraction. Typical immunoblots for syntaxin 13 are shown. Note the presence of bands in the synaptobrevin precipitants in absence, but not in presence of ATP (arrowheads). (B) Quantification of syntaxin 13 co-immunoprecipitation. Bands were quantified by densitometry (see Materials and methods); averages±s.e.m. from three independent experiments are shown. (C) The interaction between synaptobrevin and syntaxin 13 is detectable in absence of NSF activity (arrowhead). PNS fractions were incubated in presence of ATP, cytosol and NEM, to block NSF activity. Download figure Download PowerPoint We conclude that the SNARE complexes isolated by immunoprecipitation represent genuine SNARE complexes that involve SNARE motifs and that are sensitive to NSF-driven disassembly. They represent cis-complexes, as they appear in conditions where no fusion is observed—in the presence of NEM or the absence of ATP (Supplementary Figure 1; Brandhorst et al, 2006). These complexes are formed in intact membranes, and not after membrane solubilization, because they are eliminated if the NSF-mediated disassembly is allowed to proceed on intact membranes (whereas NSF activity was always inhibited in our solubilization and immunoprecipitation conditions). The relative amounts of cognate and non-cognate SNARE complexes depend on the relative concentrations of SNAREs Table I summarizes, in a qualitative manner, all of the interactions that were observed in the different preparations. It is evident that (i) not all interactions are observed in all preparations, with the complexes of synaptobrevin being the most consistent, and (ii) that in most cases, coprecipitation is observable only upon precipitation of one of the interacting partners, but not vice versa. PNS, post-nuclear supernatant. The blue circle indicates interactions observed in the early endosomal fraction from PNS of PC12 cells, the green the ones observed in PNS and the red circle the ones observed in the LS1 fraction from rat brain. The cognate interactions are enclosed by the cyan borders. To explain these seemingly discrepant results, it needs to be borne in mind that the individual SNAREs are not present in stoichiometric amounts. For instance, if synaptobrevin is 50 times more abundant than vti1a, and quantitative precipitation of synaptobrevin brings down ∼15% of the vti1a pool (Figure 2C), it is evident that quantitative immunoprecipitation of vti1a would result in the cosedimentation of only ∼0.3% of the synaptobrevin present in the starting extract, which would be difficult to discriminate from nonspecific binding to the beads. To clarify this issue, we determined the absolute amounts of the SNAREs in purified early endosomes and, for comparison, in both PC12 cell-derived PNS and a synaptic vesicle-enriched fraction isolated from rat brain synaptosomes, using purified recombinant proteins as standards (Table II; Supplementary Table 2). From the data it is evident that synaptobrevin is much more abundant than any of the other immunoprecipitated SNAREs, explaining why—relative to the starting material—no significant amounts of synaptobrevin are detectable in the immunoprecipitates of most SNAREs, whereas, conversely, all proteins except the R-SNARE VAMP4 are detectable in the immunoprecipitates of synaptobrevin. Furthermore, there are major differences between the different preparations. Syntaxin 1 is 2- to 3-fold more abundant in the synaptic vesicle-enriched fraction LS1 than in PNS or early endosomes, which explains why the cognate partner synaptobrevin is only observable in precipitates derived from LS1; the same can be stated for the non-cognate syntaxin 6. The level of VAMP4 is about an order of magnitude lower in LS1 than in early endosomes or PNS—thus, it is not detectable as interaction partner in LS1. Table 2. Quantification of the immunoprecipitated proteins in each preparation pM LS1 Early endosomes PNS Syntaxin 13 0.58±0.06 3.32±0.4 1.29±0.32 Syntaxin 6 0.46±0.07 5.44±0.19 2.59±0.09 Vti1a 0.18±0.03 0.68±0.16 0.26±0.02 Vamp4 0.015±0.005 0.43±0.03 0.28±0.04 Syntaxin 1 7.44±0.87 2.94±0.55 2.01±0.32 Synaptobrevin 127±9.2 37.89±1.65 6.77±2.99 PNS, post-nuclear supernatant. Standard curves of purified recombinant proteins were separated by SDS–PAGE alongside with different amounts of PNSs, early endosomal and LS1 fractions, followed by immunoblot analysis. The protein amounts in each preparation were calculated by interpolation. Means±s.e.m. from 3–5 independent measurements are shown. The results are presented as molar ratios in Supplementary Table 2. Thus it appears that the formation of cis-complexes is, at least in part, is governed by mass action. To substantiate these conclusions, we generated a simple Monte Carlo model describing SNARE complex formation between the two R-SNAREs synaptobrevin and VAMP4, and acceptor sites formed by the endosomal Q-SNAREs (syntaxin 13/vti1a/syntaxin 6) or by the exocytotic Q-SNAREs (syntaxin 1/SNAP-25). All SNAREs were inserted in their correct stoichiometric proportions (see Supplementary Table 2): VAMP4 (five copies), early endosomal Q-SNARE complex (eight, limited by the low copy number of vti1a), synaptobrevin (439) and the exocytotic Q-SNARE complex (34). The density of the SNAREs per unit of surface (Figure 4A) was adjusted using synaptic vesicles as model for which such quantitative data are available (Takamori et al, 2006). For simplicity, we made the assumption that the acceptor sites are permanently stable. Figure 4.Mass action determines cis-complex formation. We simulated the behavior of SNAREs in a model endosome, containing synaptobrevin, VAMP4, the early endosomal Q-SNARE acceptor complex (syntaxin 13/vti1a/syntaxin 6) and the exocytotic Q-SNARE acceptor complex (syntaxin 1/SNAP-25). We allowed the SNAREs to mix and interact for 1000 iterations, and then counted the number of both cognate and non-cognate complexes. We varied the affinity of R-SNAREs for their non-cognate complex acceptors between 1 and 1/1000. The affinity for the cognate complex was always 1. The fold difference in affinity is indicated on the x-axis. (A) The levels of the different elements are shown in brackets; the graphic description of the model is provided to give an impression of the different SNARE densities. (B) The number of complexes containing the early endosomal Q-SNARE acceptor (synaptobrevin-containing complexes, red; VAMP4-containing complexes, black). As synaptobrevin is the non-cognate partner here, its affinity decreases from left to right. The inset zooms on the first 20 points of the curves. Note that the number of synaptobrevin-containing complexes largely outnumbers that of VAMP4-containing complexes when the affinity difference is of 1–50 fold. Some non-cognate complexes form even at 1000-fold differences in affinity, when VAMP4 is still limited to only 4 of the 5 possible complexes (see also Supplementary Figure 4). (C) The number of complexes containing the exocytotic Q-SNARE acceptor. As VAMP4 is the non-cognate partner here, its affinity decreases from left to right. Note that, due to the differences in concentration, essentially no VAMP4-containing (non-cognate) complexes form, even when the affinity is 1 for both the R-SNAREs (the left-most point). Download figure Download PowerPoint The R-SNAREs were allowed to interact freely and form complexes with the QaQbQc acceptors. Collision of the cognate partners resulted always in SNARE complex formation. To test the non-cognate behavior, we varied this parameter (the probability of complex formation per collision) between 1 and 1:1000; this is equal to saying that we varied the fold difference in affinity for formation of cognate and non-cognate complexes between 1 and 1:1000. Newly formed QaQbQcR complexes persisted throughout the simulation, mimicking an ‘NSF inhibition’ situation. The model predicted fairly accurately our in vitro co-immunoprecipitation results (see the Materials and methods section for the in silico/in vitro comparison). Synaptobrevin out-competed VAMP4 when their probabilities to form complexes were comparable, until VAMP4 binds more than 150-fold more efficiently. Moreover, as the number of QaQbQc acceptor sites is higher than the number of VAMP4 molecules, some synaptobrevin-containing complexes will be able to form even when the synaptobrevin affinity is infinitesimally small (Figure 4B). Finally, VAMP4 forms only minor amounts of non-cognate complexes with syx 1/SNAP-25, even when we assume it can bind to them perfectly well (Figure 4C), showing that SNARE concentrations also determine which complexes cannot form. Thus, our data support the view that the relative amounts of different SNARE complexes in a given membrane are primarily dependent on the stoichiometric ratio between these SNAREs. In the early endosomes of PC12 cells, these proportions predict a high degree of non-cognate complexes. Surprisingly, this is also true if one assumes that there is a strong intrinsic preference for cognate SNARE pairing in the ‘cis-’configuration. Addition of soluble SNAREs onto native membranes reveals preference but not absolute specificity for cognate SNARE pairing The data described so far show that intact membranes do not contain proof-reading mechanisms selecting cognate SNAREs and/or preventing non-cognate SNAREs from forming complexes, at least not to the degree required to explain the SNARE specificity of endosome fusion and exocytosis, respectively. This finding is highly surprising, not only because it challenges co-immunoprecipitation as a means for defining cognate SNARE complexes, but also because it shows that SNARE proof-reading mechanisms must exist for an upcoming fusion event, which only operate when the SNAREs are in trans-configuration. To asse" @default.
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• W2097780147 title "The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation" @default.
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