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Vol. 19, Issue 12, 5422-5434, December 2008

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Munc18a Scaffolds SNARE Assembly to Promote Membrane Fusion

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892

Submitted May 30, 2008; Revised August 29, 2008; Accepted September 23, 2008
Monitoring Editor: Benjamin S. Glick

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Munc18a is an SM protein required for SNARE-mediated fusion. The molecular details of how Munc18a acts to enhance neurosecretion have remained elusive. Here, we use in vitro fusion assays to characterize how specific interactions between Munc18a and the neuronal SNAREs enhance the rate and extent of fusion. We show that Munc18a interacts directly and functionally with the preassembled t-SNARE complex. Analysis of Munc18a point mutations indicates that Munc18a interacts with helix C of the Syntaxin1a NRD in the t-SNARE complex. Replacement of the t-SNARE SNAP25b with yeast Sec9c had little effect, suggesting that Munc18a has minimal contact with SNAP25b within the t-SNARE complex. A chimeric Syntaxin built of the Syntaxin1a NRD and the H3 domain of yeast Sso1p and paired with Sec9c eliminated stimulation of fusion, suggesting that Munc18a/Syntaxin1a H3 domain contacts are important. Additionally, a Syntaxin1A mutant lacking a flexible linker region that allows NRD movement abolished stimulation of fusion. These experiments suggest that Munc18a binds to the Syntaxin1a NRD and H3 domain within the assembled t-SNARE complex, positioning them for productive VAMP2 binding. In this capacity, Munc18a serves as a platform for trans-SNARE complex assembly that facilitates efficient SNARE-mediated membrane fusion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
SM (Sec1p/Munc18) proteins are a class of large (68–78 kDa) evolutionarily conserved cytosolic proteins known to regulate vesicle fusion in the secretory pathway. The original SM protein unc-18 was isolated in a Caenorhabditis elegans screen for uncoordinated (unc) mutants showing defects in locomotion resulting from disruption of the nervous system (Brenner, 1974). The unc-18 orthologue in Saccharomyces cerevisiae, Sec1p, was identified later in a screen for temperature-sensitive secretion-deficient strains (Novick and Schekman, 1979; Aalto et al., 1991). Orthologues have been found in plants (KEULE; Assaad et al., 2001), invertebrates (ROP; Salzberg et al., 1993), s-Sec1 (Dresbach et al., 1998), and mammals (Munc18, also known as neuronal Sec1; Hata et al., 1993; Garcia et al., 1994; Pevsner et al., 1994b). Sec1p isoforms were subsequently discovered in each step of the secretory pathway in yeast (Novick et al., 1980; Banta et al., 1990; Ossig et al., 1991; Cowles et al., 1994), and higher organisms (Weimer and Richmond, 2005). In mammals, the SM protein Munc18 has been implicated in exocytosis (Verhage et al., 2000; Fisher et al., 2001; Graham et al., 2004; Ciufo et al., 2005; Schutz et al., 2005; Burgoyne and Morgan, 2007). The three Munc18 isoforms: Munc18a (Munc18-1), Munc18b (Munc18-2), and Munc18c (Munc18-3) display tissue-specific expression, with Munc18a being localized almost exclusively to brain (Pevsner et al., 1994b; Weimer and Richmond, 2005).

SM proteins have been shown to function biochemically by interacting with SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins (Sollner et al., 1993). Most SNAREs are tail-anchored proteins that specifically distribute along the secretory pathway. They assemble in a combinatorial manner at each vesicular trafficking step in the secretory pathway to mediate membrane fusion (Jahn and Scheller, 2006). SNARE proteins are operationally divided into those primarily present on the transport vesicle (v-SNAREs) and target plasma membrane (t-SNAREs; Sollner et al., 1993). SNARE proteins have two defined functions that are distinct but interrelated. Their first role is to provide the final layer of proofreading to assure that the ensuing fusion event is compartmentally correct (McNew et al., 2000). Specific pairing of v- and t-SNAREs fulfills this proofreading function. Their second role is to provide mechanical energy to drive the fusion reaction, which is achieved by the SNARE complex assembly process (Weber et al., 1998). Each SNARE protein contains an 60-residue SNARE motif that consists of a characteristic heptad repeat sequence with a strong propensity to form coiled-coils (Weimbs et al., 1997). Membrane fusion is driven by the "zippering" of SNARE motifs in the appropriate combination to provide the mechanical force necessary for fusion of phospholipid bilayers (Weber et al., 1998; Melia et al., 2002).

The association of SM proteins with Syntaxin family proteins has been well documented. Protein expression levels of Syntaxin1a and Munc18a have been closely linked (Voets et al., 2001; Arunachalam et al., 2008), and in vivo data suggest Munc18a stabilizes and traffics Syntaxin1a to the plasma membrane in some cell types (Medine et al., 2007). Biochemical work also has shown that Munc18a interacts strongly with the free t-SNARE Syntaxin1a (Pevsner et al., 1994a), which stabilizes the "closed" conformation of Syntaxin1a within the cavity of the arch-shaped Munc18a molecule (Misura et al., 2000; Yang et al., 2000). This binary Munc18a/Syntaxin1a interaction is thought to prevent the cognate t-SNARE SNAP25 from binding to Syntaxin1a, thereby blocking formation of the assembled t-SNARE complex necessary for vesicle fusion (Yang et al., 2000). However, recent work with SNAREs in native plasma membranes provides evidence that SNAP25 can enter into a t-SNARE complex, whereas Syntaxin1a is bound to Munc18a as a heterodimer (Zilly et al., 2006), possibly aided by arachidonic acid (Connell et al., 2007) or Munc13 (Hammarlund et al., 2007). The strong affinity between Syntaxin1a and Munc18a derives from the extensive interactions within Syntaxin1a, including an extreme N-terminal peptide, the N-terminal regulatory domain (NRD, also called the Habc domain) and H3 "core" domains and Munc18a (Misura et al., 2000; Burkhardt et al., 2008). Some of these interactions also have been observed between other SM proteins such as Munc18c, Sly1p, and Vps45p and their cognate Syntaxins (Dulubova et al., 2002; Yamaguchi et al., 2002; D'Andrea-Merrins et al., 2007).

Other modes of SM protein/SNARE interactions have also been documented. The yeast SM protein Sec1p binds weakly if at all to Sso1p, the yeast Syntaxin1a homolog. Instead, it binds strongly to the t-SNARE complex (Scott et al., 2004) as well as the fully assembled ternary SNARE complex (Carr et al., 1999; Scott et al., 2004). Sly1p was shown to bind non-Syntaxin t-SNAREs (Peng and Gallwitz, 2004). Vps45p, a yeast SM protein involved in transport from the Golgi to late endosomes (Cowles et al., 1994), has been shown to bind the yeast v-SNARE Snc1p as well as the cognate Syntaxin Tlg2p (Carpp et al., 2006), suggesting that SM proteins could associate with v-SNAREs as well as t-SNAREs.

Although SM proteins have been studied extensively in many systems, their suggested functions have been contradictory (reviewed in Gallwitz and Jahn, 2003; Toonen and Verhage, 2003; Weimer and Richmond, 2005; Burgoyne and Morgan, 2007). SM proteins have been proposed to play both inhibitory and stimulatory roles in the fusion process (Wu et al., 1998, 2001; Verhage et al., 2000; Yang et al., 2000; Scott et al., 2004). Biochemical evidence that Munc18a could block t-SNARE complex formation led to the hypothesis that Munc18a inhibits vesicle fusion. This idea was bolstered by work suggesting that overexpression of the Drosophila melanogaster SM protein ROP (ras opposite; Salzberg et al., 1993), which exhibits 65% sequence similarity with Munc18a, inhibited neurotransmission (Wu et al., 1998). However, the results of several other in vivo experiments indicated that Munc18a plays a positive role in neurotransmission. Mouse Munc18a knockouts resulted in a lethal phenotype due to severe neurodegeneration immediately after birth (Verhage et al., 2000), although brain development was unaffected, pointing to an absolute requirement for Munc18a in neurosecretion. Furthermore, elimination of Munc18a in mouse chromaffin cells reduced Ca²⁺-dependent exocytosis of large dense core vesicles by a factor of 10 (Voets et al., 2001). Studies of SM proteins in other organisms have also pointed toward a positive rather than negative role for SM protein function. Loss of function alleles of Sec1p in S. cerevisiae or ROP in D. melanogaster result in lethal phenotypes (Novick et al., 1980; Harrison et al., 1994; Schulze et al., 1994). Although loss of the Munc18a homolog in C. elegans (unc18) is not lethal, it leads to severe impairment of synaptic transmission (Weimer et al., 2003).

To elucidate the function of SM proteins, we previously focused on Sec1p, the yeast homolog of Munc18a. Sec1p was shown to bind strongly to the preassembled yeast t-SNARE and ternary SNARE complexes, and that binding directly stimulated fusion of yeast SNAREs in an in vitro fusion assay (Scott et al., 2004). This original observation was supported further by a recent study showing that Munc18a also directly promotes membrane fusion in vitro and that its stimulatory effect is specific to the neuronal SNAREs Syntaxin 1A, SNAP25, and VAMP2 (Shen et al., 2007).

Here, we used mutational and domain swap analyses to dissect further the interactions of Munc18a with the neuronal SNARE complex. We found that Munc18a interacts directly with the assembled Syntaxin1a/SNAP25 t-SNARE complex and minimally with the v-SNARE VAMP2. Munc18a stimulates neuronal SNARE-mediated fusion by about twofold in an in vitro fusion assay, and an ionic interaction between Munc18a and the Syntaxin1a NRD contributes to the accelerated fusion. The stimulatory effect of Munc18a can essentially be abolished by replacing the SNARE domain of Syntaxin with the homologous yeast Sso1 SNARE domain. Interestingly, deletion of the flexible linker region between the NRD and H3 domain of Syntaxin1a (residues 159–182) substantially reduced the positive effect of Munc18a. These results suggest that Munc18a recognizes the Syntaxin1a H3 domain within the assembled t-SNARE complex in addition to known contacts with the NRD. Binding of Munc18a to the assembled t-SNARE complex may place the NRD in a conformation that favors VAMP2 binding and subsequently accelerates SNARE-mediated membrane fusion.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cloning
The following SM and SNARE Plasmid constructs were created.

Untagged Syntaxin1a (pJM537). An untagged version of Rat Syntaxin1a was cloned by PCR of pTW34 (Parlati et al., 1999) using oligos 426 (5'-CGC ATA TGA AGG ACC GAA CCC AGG AGC-3') and 427 (5'-GCC TCG AGC TAT CCA AAG ATG CCC CCG ATG G-3'). The PCR product was cloned into pCR-Blunt II-TOPO using a Zero Blunt PCR cloning kit (Invitrogen, Carlsbad, CA), producing pJM525. The clone was verified by sequencing. The gene was cut out of pJM525-2 by restriction enzyme digest with NdeI and XhoI, and the resulting fragment was ligated into pETDuet-1 (EMD Biosciences, La Jolla, CA) cut with the same enzymes.

Syntaxin1aNRD (1-178)/Sso1NRD (pJM579). A His₈-Syntaxin1a NRD (Rat)/Sso1 NRD (Yeast) chimera was cloned by PCR from the yeast expression vector pJM413 with oligos 301 (5'-GCG AAT TCA TGA AGG ACC GAA CCC AGG-3') and 103 (5'-GAG ATA TCC TCG AGT TAA CGC GTT TTG ACA ACA GCT GGG-3'). The PCR product was digested with EcoRI and XhoI and then ligated into pET24a(+) (EMD Biosciences) cut with the same enzymes. The clone was verified by sequencing.

Syntaxin1a 159-182 (pJM590). An untagged Syntaxin1a 159-182 was cloned by PCR of pTW34 with oligos 426 (5'-CGC ATA TGA AGG ACC GAA CCC AGG AGC-3') and 485 (5'-CCG AAT TCC CGG CCA GTG ATC TCC AGC-3'), producing the sequence coding for amino acids 1-158 (Syntaxin1a NRD), and PCR of pTW34 with oligos 427 (5'-GCC TCG AGC TAT CCA AAG ATG CCC CCG ATG G-3') and 486 (5'-GCA CAT ATG GAA TTC ATG GAC TCC AGC ATC TCG-3'), producing the sequence coding for amino acids 183–289 (Syntaxin H3 domain). The H3 domain fragment was cut with EcoRI and XhoI and ligated into pET24a(+) cut with the same enzymes, producing pJM583. The NRD fragment (1-158) was cut with Nde1 and EcoRI and ligated into pJM583 cut with the same enzymes, producing pJM590. The resulting clone was verified by sequencing.

Syntaxin1a 159-182-H₆ (pJM595). The sequence coding for His₆-Syntaxin1a 159-182 was cut out of pJM590 by restriction enzyme digest with MscI and XhoI. The resulting fragment was ligated into pTW12 (Weber et al., 1998) cut with the same enzymes.

Munc18a-H₆ R39E (pJM550). A site-directed mutagenesis kit (Stratagene, La Jolla, CA; QuikChange II) was used to create Munc18a-His₆ R39E. PCR of pJM537 as template was performed with oligos 453 (5'-GTG GAC CAG TTA AGC ATG GAG ATG CTG TCT TCC TGC TG-3') and 454 (5'-CAG CAG GAA GAC AGC ATC TCC ATG CTT AAC TGG TCC AC-3') in order to create the mutation. The resulting PCR product was sequenced to confirm the mutation.

Munc18a-H₆ E59R (pJM551). A site-directed mutagenesis kit (Stratagene; QuikChange II) was used to create Munc18a-His₆ E59R. PCR of pJM537 as template was performed with oligos 447 (5'-GAG GGG ATC ACA ATT GTG AGG GAT ATC AAC AAG CGC CG-3') and 448 (5'-CGG CGC TTG TTG ATA TCC CTC ACA ATT GTG ATC CCC TC-3') in order to create the mutation. The resulting PCR product was sequenced to confirm the mutation.

Munc18a-H₆ T574D (pJM563). A site-directed mutagenesis kit (Stratagene; QuikChange II) was used to create Munc18a-H₆ T574D. PCR of pJM537 as template was performed with oligos 471 (5'-GAT CCA CGC ACA TTC TCG ACC CAC AGA AAC TGC TGG-3') and 472 (5'-CCA GCA GTT TCT GTG GGT CGA GAA TGT GCG TGG ATC-3') in order to replace T574 with an aspartic acid. The resulting PCR product was sequenced to confirm the mutation.

Recombinant Protein Production and Reconstitution
The following SM proteins were expressed and purified.

Munc18a. Munc18a-His₆ (Rattus norvegicus) was produced by expressing pJM546 (plasmid received from Dr. Jingshi Shen, University of Colorado at Boulder) in BL21 (DE3) Escherichia coli (Stratagene). Cells were grown at 37°C in 4 l of SuperBroth (Teknova, Half Moon Bay, CA) to an OD600 of 0.75 while shaking at 200 rpm in a baffled 6-l flask. Protein expression was induced with 1 mM IPTG while shaking 3 h at 37°C at 200 rpm. Cells were pelleted by centrifugation and then immediately resuspended in 50 ml of buffer A150 (25 mM HEPES, pH 7.4, 150 mM KCl, 10% glycerol, and 1 EDTA-free protease inhibitor tablet; Roche, Indianapolis, IN). Cells were passed through an Emulsiflex-C5 High Pressure Homogenizer (Avestin, Ottawa, ON, Canada) and lysed by pressure. Cellular debris was removed by centrifugation at 186,000 x g_max for 1 h at 4°C. Cell extract was filtered at 4°C first through a sterile 1.2 µM filter (Millipore, Bedford, MA) and then through a sterile 0.45-µM filter (Millipore). Extract was then passed over a HiTrap HP Ni²⁺-chelating column (GE Healthcare, Waukesha, WI) in an ÄKTA Prime chromatography system (Amersham Biosciences, Piscataway, NJ) to bind Munc18a-H₆. The column was washed with 10 column volumes of buffer A150 (25 mM HEPES, pH 7.4, 150 mM KCl, 10% glycerol, and 2 mM β-mercaptoethanol [BME]). Protein was eluted in 20 column volumes with a linear gradient of 20 to 500 mM imidazole in buffer A150. Peak fractions were pooled and then immediately dialyzed against 4 l of buffer A500 (25 mM HEPES-KOH, 500 mM KCl, 10% glycerol, and 1 mM DTT, pH 7.4) overnight at 4°C in a microdialysis chamber (Gilson, Worthington, OH) to exchange imidazole with KCl. Protein was then dialyzed 4 h at 4°C against 1 l of buffer A350 (25 mM HEPES, 350 mM KCl, 10% glycerol, and 1 mM DTT), followed by dialysis against 1 l buffer A200 (25 mM HEPES, 200 mM KCl, 10% glycerol, and 1 mM DTT) for 4 h at 4°C to reduce the KCl concentration. Protein was aliquoted, flash frozen in liquid nitrogen, and stored at –80°C. Preps were quantitated with Amido Black assays (Schaffner and Weissmann, 1973), and yields ranged from 9.44 to 12.71 mg/ml (4 ml).

SM Protein Munc18a Mutants. Munc18a-His₆ T574D, Munc18a-His₆ E59R, and Munc18a-His₆ R39E were all expressed and purified as described above for Munc18a-His₆. Yields were (15.46 mg/ml, T574D; 8.98 mg/ml, E59R; and 8.64 mg/ml, R39E) as determined by Amido Black assays.

The following t-SNARE proteins were expressed and purified.

Syntaxin1a/SNAP25b. Syntaxin1a (rat) and His₆-SNAP25b (mouse) were coexpressed from the dicistronic plasmid pTW34. Eight liters of E. coli [BL21(DE3)] were grown at 37°C in SuperBroth to an OD600 of 0.70. Protein expression was induced with 0.25 mM IPTG for 4 h at 37°C. Cells were pelleted, resuspended in buffer A400, pH 7.4 (25 mM HEPES, 400 mM KCl, 10% glycerol, and 2 mM BME), and then lysed as described for Munc18a-His₆ in the presence of 1% Triton X-100. Extract was clarified, filtered, and passed over an Ni²⁺-chelating column as described for Munc18a-His₆. The column was washed with 10 column volumes of buffer A400 containing 1% Triton X-100. Triton X-100 was exchanged for n-octyl-β-D-glucopyranoside (OG) by washing with 25 column volumes of buffer A100 (25 mM HEPES-KOH, 100 mM KCl, 10% glycerol, and 2 mM BME, pH 7.4) containing 1% OG. Protein was eluted in 20 column volumes with a linear gradient of 20 to 500 mM imidazole in buffer A100 (25 mM HEPES, 100 mM KCl, 10% glycerol, 2 mM BME, and 1% OG). Protein was aliquoted as described above and quantitated with an Amido Black assay. Yields ranged from 4.05 to 5.55 mg/ml. The resulting t-SNARE complex was reconstituted by detergent dilution and dialysis as described (Scott et al., 2003).

Syntaxin1a/Sec9c. Untagged Syntaxin1a (rat) and H₈-Sec9c were coexpressed from pJM537 and pJM418 and purified as described above for Syntaxin1a/H₆-SNAP25b. However, protein was lysed in buffer A400, washed with A400 + 1% TX-100 and A400 + 1% OG, and then eluted with buffer A400 + 1% OG in order to help prevent precipitation as observed from trials eluting with lower [KCl]. Yield was 3.02 mg/ml as determined by an Amido Black assay. The resulting mixed t-SNARE complex was reconstituted as described for Syntaxin1a/His₆-SNAP25b.

Syntaxin1a NRD-Sso1NRD/Sec9c. The His₈-Syntaxin1a NRD-Sso1NRDchimera/His₈-Sec9c t-SNARE complex was expressed separately from pJM579 and pJM418. pJM418 was purified as described previously (Liu et al., 2007a) with a yield of 4.5 mg/ml as determined by an Amido Black assay. His₈-Syntaxin1a NRD-Sso1NRD was reconstituted by itself as described for Syntaxin1a/His₆-SNAP25b. The resulting proteoliposomes were mixed with a threefold molar excess of His₈-Sec9c overnight at 4°C and then refloated 4 h at 4°C at 218,500 x g_max in 0%/30%/40% density step gradients to remove unbound Sec9c.

Syntaxin1a 159-182/SNAP25b. The Syntaxin1a159-182/His₆-SNAP25b t-SNARE complex was coexpressed from pJM595 and pFP247 (Parlati et al., 1999) and purified as described before for Syntaxin1a/His₆-SNAP25b. Yield was 1.19 mg/ml as determined by an Amido Black assay. Syntaxin1a159-182/His₆-SNAP25b t-SNARE complex was reconstituted as described for Syntaxin1a/His₆-SNAP25b.

Sso1p/Sec9c. His₈-Sso1p (yeast) and His₈-Sec9c (yeast) were expressed and purified separately from pJM88–1 and pJM418, respectively, as previously described (McNew et al., 2000; Liu et al., 2007a). Yields were 2.3 mg/ml for His₈-Sso1p and 4.5 mg/ml for His₈-Sec9c. His₈-Sso1p was reconstituted as described for Syntaxin1a/H₆-SNAP25b, then mixed with a threefold molar excess of His₈-Sec9c, and refloated as described for His₈-Syntaxin1a NRD-Sso1NRD/His₈-Sec9c.

Syntaxin1a 159-182. Syntaxin1a159-182-His₆ was expressed from pJM595 as described for Syntaxin1a/His₆-SNAP25b, except that 0.5% glucose was added to the growth medium. Yield was 2.26 mg/ml as determined by an Amido Black assay. Syntaxin1a159-182-His₆ was reconstituted as described for His₈-Sso1p (McNew et al., 2000).

The following v-SNARE proteins were expressed and purified.

VAMP2. VAMP2-His₆ (mouse) was expressed and purified from pTW38 as described previously (Parlati et al., 1999). Yield was 6.41 mg/ml as determined by an Amido Black assay. VAMP2-His₆ was reconstituted into labeled donor proteoliposomes as described (Scott et al., 2003).

Snc1p. Snc1p-His₆ (pJM90–1) was expressed and purified as described previously (McNew et al., 2000). Yield was 4.5 mg/ml. Snc1p-His₆ was reconstituted as described for VAMP2-His₆.

Reconstitution by Detergent Dilution and Dialysis
SNARE proteins were reconstituted into proteoliposomes by detergent dilution and dialysis as previously described (Scott et al., 2003). Protein in 1% OG (final volume 500 µl for t-SNARE acceptor proteoliposomes, 300 µl for v-SNARE donor proteoliposomes) was mixed with a dried film of 85 mol% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/15 mol% 1,2-dioleoylphosphatidylserine (DOPS) at RT for 15 min and then diluted with the appropriate buffer to reduce OG below its critical micellar concentration (CMC; Scott et al., 2003). OG was then removed from the resulting proteoliposomes by dialysis for 16 h at 4°C in a microdialysis flow chamber (Gilson). Proteoliposomes were then separated from unincorporated lipid and protein by floatation for 4 h at 4°C at 48,000 rpm (218,500 x g_ave) in a Beckman SW-55Ti rotor (Fullerton, CA) through a 40%/30%/0% discontinuous density step gradient made of Accudenz (Accurate Chemical and Scientific, Westbury, NY) dissolved in buffer + 1 mM DTT. Proteoliposomes were harvested (400 µl t-SNARE, 75 µl v-SNARE) at the 0%/30% gradient interface. The amount of protein incorporated was determined by Amido Black assays.

In Vitro Fusion Assay
Proteoliposomes to be fused were mixed in 96-well Fluoronunc Polysorp plate (Nunc, Naperville, IL) strips, covered in aluminum foil, and incubated on ice for 3 h. A 3-h preincubation period was empirically determined to produce the maximum stimulation of fusion (data not shown). Ninety-six-well plate strips then were placed in a fluorescent plate reader (Floroskan II, MTX Lab Systems, Vienna, VA) preheated to 37°C. Acceptor (t-SNARE) and donor (v-SNARE) proteoliposomes were fused 2 h at 37°C as described previously (Scott et al., 2003) with readings taken every 2 min. Ten microliters of 2.5% (wt/vol) n-dodecylmaltoside detergent was added at 2 h to produce maximum NBD fluorescence. NBD fluorescence was normalized to percent of maximum fluorescence.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Munc18a Stimulates In Vitro Fusion of Syntaxin1a/SNAP25b and VAMP2
Yeast Sec1p was shown to strongly and positively regulate membrane fusion in vitro (Scott et al., 2004), and Munc18a appears to promote membrane fusion in a similar manner (Shen et al., 2007). We produced recombinant Munc18a-H₆ in E. coli to further characterize in detail its effect on in vitro fusion with the neuronal t-SNAREs Syntaxin1a/H₆-SNAP25b and v-SNARE VAMP2-H₆. Unlike yeast Sec1p, Munc18a-H₆ was very well expressed (12.5 mg/l of culture) and largely stable at high concentrations (140 µM) under neutral pH conditions at moderately high salt concentrations (> 200 mM KCl, see Materials and Methods for details; however, it should be noted that the ability of Munc18a to stimulate SNARE-mediated fusion is highly salt sensitive; see Supplemental Figure S5). The ability to produce Munc18a-H₆ at high concentrations allowed for the addition of soluble Munc18a-H₆ directly to a standard fusion reaction.

For the in vitro fusion assays, full-length Syntaxin1a and H₆-SNAP25b were coexpressed in E. coli and reconstituted as a functional t-SNARE complex into acceptor liposomes containing 85 mol% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/15 mol % 1,2-dioleoylphosphatidylserine (DOPS). Fluorescently labeled donor v-SNARE liposomes containing POPC, DOPS, and the headgroup labeled lipids [N-(7-nitro-2,1,3-benzoxadiazole-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine (NBD-DPPE) and N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl phosphatidylethanolamine (Rh-DPPE)] were produced with recombinant VAMP2-H₆. Fusion is measured as an increase in NBD fluorescence when unlabeled acceptor membrane merges with the fluorescently labeled donor membrane, relieving quenched NBD fluorescence due to fluorescence resonance energy transfer (FRET) between the two fluorescent lipids (Weber et al., 1998; Scott et al., 2003). Syntaxin1a/SNAP25b and VAMP2 proteoliposomes were mixed in the presence or absence of 20 µM Munc18a-H₆ for 3 h at 4°C, and the effects on SNARE-mediated fusion were examined (Figure 1A).

View larger version (13K): [in this window] [in a new window]   Figure 1. Soluble Munc18a Strongly Stimulates in vitro fusion. (A) Kinetic fusion graph of unlabeled acceptor neuronal t-SNARE (Syntaxin1a/SNAP25b) proteoliposomes fused with fluorescently labeled donor neuronal v-SNARE (VAMP2) proteoliposomes in the presence () or absence () of 20 µM Munc18a. Background fusion (solid line) was measured by the addition of the cytoplasmic domain of VAMP2 (CDV) in a 1.6-fold molar excess of t-SNARE protein to inhibit fusion. The inclusion of 10 µM Munc18a in the inhibited fusion reaction serves as an additional control for background fusion. Both proteoliposome populations and Munc18a were preincubated 3 h at 4°C and then fused 2 h at 37°C in a standard fusion reaction. Data are represented as percent (%) of maximum fluorescence versus time. Fusion reactions contained 45 µl of Syntaxin1a/SNAP25 proteoliposomes (55.4 µg, 0.962 nmol protein, 44 nmol lipid) and 5 µl of VAMP2 proteoliposomes (2.6 µg, 0.189 nmol protein, 5.3 nmol lipid). All fusion reactions were brought up to a final volume of 60 µl with Munc18a, buffer A100, CDV, or a combination of the three. (B) Titration of soluble Munc18a into in vitro fusion reactions. Increasing amounts (µM) of Munc18a were added to a series of independent fusion reactions. Background fusion measured by the addition of CDV to a fusion reaction in the presence of 10 µM Munc18a was subtracted from the endpoint fusion of each reaction, yielding net endpoint fusion. Net fusion (% of maximum fluorescence) of each reaction at 120 min was plotted against [Munc18a] (). The titration curve was fit with a sigmoidal equation in the Kaleidagraph package (Abelbeck Software, Reading, PA). Saturation of increase in fusion occurs at 20 µM Munc18a. (C) Net fold stimulation in initial rate and final extent of in vitro fusion resulting from addition of 20 µM Munc18a as described in A. Endpoint fusion (% maximum fluorescence) for 14 independent fusion reactions of Syntaxin1a/SNAP25 and VAMP2 in the presence and absence of 20 µM Munc18a was averaged. Endpoint fusion (% maximum fluorescence) of seven independent fusion reactions of Syntaxin1a/SNAP25, CDV, VAMP2, and 10 µM Munc18a was averaged to determine the average background signal. The average background signal was subtracted from the average endpoint fusion of reactions in the presence and absence of 20 µM Munc18a to yield net stimulation. Error bars, SEM. The rates of fusion between the 6-min (after temperature change from 4 to 37°C) and 10-min mark were calculated in fusion reactions of Syntaxin1a/SNAP25/VAMP2 in the presence and absence of 20 µM Munc18a by linear curve fits. The initial rate increased from 0.39%/min in reactions without Munc18a (, left) to 1.09%/min in reactions with 20 µM Munc18a (, right), an improvement of 2.8-fold.

The increased fusion mediated by Munc18a is saturable and reaches a maximum of 1.8-fold at 20 µM Munc18a under these conditions (Figure 1, B and C). When the extent of fusion at 120 min is adjusted to account for fusion during the preincubation period, a more robust stimulation is observed (mean = 2.2-fold, n = 14, data not shown). The stimulation of fusion measured at 120 min is mirrored by a similarly significant improvement in the initial rate of fusion when Munc18a-H₆ is present. The initial rate (measured as a percent of maximum fusion per minute) increased by 2.8-fold in the presence of Munc18a (Figure 1C).

The observed stimulation of fusion could be the result of Munc18a interaction with any of the participating SNAREs. We next determined if Munc18a was interacting preferentially with one of the SNARE subcomplexes by mixing Munc18a for 3 h with either t-SNARE or v-SNARE proteoliposomes independently and then adding the other proteoliposome population immediately before initiation of fusion at 37°C. Preincubation of Munc18a with t-SNARE (Figure 2, ) or v-SNARE () proteoliposomes alone with 20 µM Munc18a-H₆ resulted in no stimulation of fusion, with initial rate and final extent of fusion essentially equal to SNARE-only control reactions (Figure 2, ). As expected, inclusion of all components during preincubation resulted in robust stimulation of fusion () that was completely dependent on SNARE complex formation as shown by inhibiting a fusion reaction with the cytoplasmic domain of VAMP2 (20 µM CDV, solid line), which agrees with the findings of others (Shen et al., 2007). These results suggest that both the t-SNARE complex and the v-SNARE communicate with Munc18a during the fusion reaction.

View larger version (15K): [in this window] [in a new window]   Figure 2. Preincubation of Munc18a with both Syntaxin1a/SNAP25 and VAMP2 is required for stimulation of fusion. Strong stimulation was observed when t-and-v-SNAREs were preincubated for 3 h with 20 µM Munc18 () compared with SNARE-only reactions with preincubation (). However, no stimulation was observed when Syntaxin1a/SNAP25 proteoliposomes were preincubated with 20 µM Munc18a with VAMP2 proteoliposomes added immediately before fusion (). Conversely, when VAMP2 proteoliposomes were preincubated with 20 µM Munc18a with Syntaxin1a/SNAP25 proteoliposomes added immediately before fusion (), no significant stimulation of fusion above that of the SNARE-only control () was observed. All reactions contained 45 µl Syntaxin1a/SNAP25 acceptor proteoliposomes (55.4 µg, 0.962 nmol protein, 44 nmol lipid), 5 µl VAMP2 donor proteoliposomes (2.6 µg, 0.189 nmol protein, 5.3 nmol lipid), and were brought up to a final volume of 60 µl with Munc18a and buffer A100. Proteoliposomes were preincubated 3 h at 4°C then fused 2 h at 37°C as described in Figure 1.

Second, we formed potential t-SNARE complex/Munc18a complexes in detergent solution that subsequently were reconstituted as units. This method was used in our previous work with Sec1p and the yeast t-SNAREs Sso1p/Sec9c (Scott et al., 2004). Binding in the absence of lipid was accomplished by mixing Syntaxin1a/H₆-SNAP25 protein in a 0.75% OG detergent solution with a threefold molar excess of Munc18a-H₆ at 4°C for 4 h. The resultant protein mixture was reconstituted into liposomes and floated through a density step gradient to remove unbound Munc18a. Significant but substoichiometric binding of Munc18a to the t-SNARE complex was observed (Figure 3A).

View larger version (13K): [in this window] [in a new window]   Figure 3. Munc18a functionally interacts with the assembled t-SNARE complex. (A) Munc18a binds the assembled t-SNARE complex. Syntaxin1a/H6-SNAP25 protein in 1% OG was mixed with a threefold molar excess of Munc18a for 4 h at 4°C at a final [KCl] of 133 mM and final OG of 0.8%, and the mixture was reconstituted into acceptor liposomes by the standard detergent dilution and dialysis method. Proteoliposomes were run on a Novex NuPage 10% Bis-Tris gel (Encinitas, CA), and binding of Munc18a was resolved by staining the gel with Coomassie blue. Ten microliters of sample was loaded per lane. Lane 1, unlabeled Syntaxin1a/SNAP25 proteoliposomes; lane 2, unlabeled Syntaxin1a/SNAP25/Munc18a proteoliposomes; lane 3, SNARE-free liposomes prepared in the presence if Munc18a. Munc18a binds the assembled t-SNARE complex, although apparently in substoichiometric amounts. (B) Functional consequences of Munc18a binding to Syntaxin1a/SNAP25. Munc18a bound to the preassembled t-SNARE complex stimulates fusion to the same extent as soluble 20 µM Munc18a added to a fusion reaction of Syntaxin1a/SNAP25 acceptor and VAMP2 donor proteoliposomes and was further stimulatable by addition of soluble 20 µM Munc18a to the fusion reaction. Forty-five microliters of Munc18a prebound to the t-SNARE complex () was preincubated 3 h at 4°C with VAMP2 donor liposomes in the presence () or absence of 20 µM soluble Munc18a and then was fused for 2 h at 37°C. Fusion of Munc18a prebound to Syntaxin1a/SNAP25 was stimulated to essentially the same extent as fusion of Syntaxin1a/SNAP25 and VAMP2 in the presence of 20 µM soluble Munc18a (not shown) compared with a control reaction without Munc18a (). A roughly 1.8-fold stimulation of fusion above the SNARE-only control reaction was observed for Munc18a prebound to the t-SNARE complex and for a reaction with 20 µM Munc18a added in solution. Addition of soluble 20 µM Munc18a to a reaction of Munc18a prebound to Syntaxin1a/SNAP25 and VAMP2 resulted in a further 1.56-fold stimulation (2.8-fold above SNARE-only control). Background fusion was measured by inhibiting a fusion reaction (solid line) with 20 µM CDV.

Evidence that Vps45p binds the v-SNARE Snc1p (Carpp et al., 2006) led us to investigate Munc18a/VAMP2 interactions. Detectable but substoichiometric binding of Munc18a to VAMP2 was observed by both binding and reconstituting in detergent solution (data not shown) and binding to preformed VAMP2 proteoliposomes (Supplemental Figure S4). His₆-VAMP2 could also be copurified with untagged Munc18a as described above for the t-SNARE complex, although to a lesser extent (data not shown).

Munc18a Prebound to the Assembled t-SNARE Complex Stimulates Fusion
We next investigated the functional consequences of Munc18a binding to the assembled t-SNARE complex. When Munc18a and Syntaxin1a/SNAP25 were reconstituted in detergent solution as a unit (Figure 3A) and used as acceptor liposomes in a fusion assay, improved fusion was observed relative to t-SNARE complexes without bound Munc18a (Figure 3B, , vs. ). The stimulation observed by bound Munc18a still required prior preincubation with v-SNARE proteoliposomes. However, the addition of 20 µM soluble Munc18a to the fusion reaction containing Munc18a bound Syntaxin1a/SNAP25 acceptor proteoliposomes resulted in further stimulation (Figure 3B, ). This increased stimulation is likely due to the substoichiometric binding of Munc18a to the t-SNARE complex. These data confirm that the Munc18a bound to the t-SNARE complex is a functional intermediate.

Ionic Interactions are Required for Munc18a–mediated Stimulation of Fusion
Fully zipped SNARE complexes form a four-helix bundle with a highly charged surface that may interact with regulatory proteins (Sutton et al., 1998). Inspection of the Munc18a/Syntaxin1a crystal structure also suggests that ionic interactions may contribute to this interaction (Misura et al., 2000). To test whether Munc18a-mediated fusion stimulation depends on ionic interactions with SNAREs, potassium chloride was titrated into independent fusion reactions with 20 µM Munc18a to determine the effect of increased ionic strength. Interestingly, Munc18a-mediated fusion stimulation exhibited a dramatic salt sensitivity, as an increase from 100 to 200 mM KCl essentially abolished stimulation of fusion (Supplemental Figure S5).

Molecular modeling of the Munc18a/Syntaxin1a heterodimer indicated that ionic interactions may exist between arginine 114 in Syntaxin1a and glutamate 59 in Munc18a as well as glutamate 234 in Syntaxin1a and arginine 39 in Munc18a. To test whether those two ionic interactions are required for Munc18-mediated stimulation, site-directed mutagenesis was used to mutate Munc18a R39 and Munc18a E59 to residues carrying the opposite charge (ER and RE) in order to destroy the postulated salt bridges. Munc18a R39E and Munc18a E59R mutants were cloned, expressed, and purified as described for wild-type Munc18a. The effects of these mutations were assayed by adding 20 µM of either Munc18a R39E or Munc18a E59R to fusion reactions of Syntaxin1a/SNAP25b and VAMP2 and by comparing initial rates and endpoint fusion to reactions with same neuronal SNAREs and 20 µM wild-type Munc18a. Munc18a R39E showed no significant deficit in initial rate of fusion, although endpoint fusion was slightly impaired, but not statistically significant (Figure 4A, ). In contrast, the initial rate of fusion of Munc18a E59R was 40% slower than wild type (2.83- vs. 4.46-fold) and stimulation of endpoint fusion was impaired by 25% (1.56- vs. 2.02-fold; Figure 4B).

View larger version (14K): [in this window] [in a new window]   Figure 4. Ionic interactions with SNAREs are important for Munc18a-mediated stimulation of fusion. (A) Representative kinetic fusion graph of Syntaxin1a/SNAP25b proteoliposomes fused with VAMP2 proteoliposomes in the absence () or presence of 20 µM wild-type or mutant (R39E, E59R, and T574D) Munc18a. The fusion assay was performed as described in Figure 1A. Background fusion (solid line) was measured by inhibiting a standard fusion reaction with 20 µM CDV. Wild-type Munc18a () produced about a twofold stimulation of endpoint fusion at 120 min compared with a SNARE-only control reaction (). Little deviation from wild-type Munc18a was observed with the Munc18a phosphomimetic T574D (), whereas a small deficit in endpoint fusion was observed with Munc18a R39E (), and a more noticeable deficit in endpoint fusion and initial rate was detected with Munc18a E59R (). (B) Quantification of differences in net fusion and initial rate of fusion resulting from Munc18a mutations. Top panel, histogram of differences in net fusion at 120 min. Endpoint net fusion from multiple independent fusion reactions was calculated by subtracting endpoint background signal values from endpoint values of SNARE-only reactions and reactions in the presence of Munc18a. Net fold stimulation was then calculated by dividing net fold stimulation of reactions with wild-type or mutant Munc18a by net fold fusion of equivalent SNARE-only reactions. All reactions contained 45 µl Syntaxin1a/SNAP25b proteoliposomes and 5 µl VAMP2 proteoliposomes, and wild-type or mutant Munc18a was added to final concentration of 20 µM. All reactions were mixed to a final volume of 60 µl. T-SNARE proteoliposome protein:lipid ratios ranged from 1:100-1:200, and v-SNARE proteoliposome protein:lipid ratios ranged from 1:30 to 1:80 as determined by Amido Black assays and lipid recovery after reconstitution as determined by scintillation count of 3H incorporated into the lipid. Wild-type Munc18a produced a 2.02-fold stimulation of net fusion, whereas Munc18a E59R showed an 25% impairment (1.56-fold stimulation). An 10% deficit in stimulation was observed with Munc18a R39E (1.81-fold stimulation), whereas the phosphomimetic Munc18a T574D showed slightly increased endpoint fusion (2.16-fold stimulation). Results with Munc18a E59R were statistically significant as determined by a paired Student's t test with p < 0.0001. Although small differences in initial rate and endpoint fusion were consistently observed with Munc18a R39E and Munc18aT574D, these differences were not statistically significant by a paired Student's t test. Error bars, SEM. Bottom, histogram of differences in initial rate (6–10 min) of fusion among the Munc18a mutants. Net fusion (% max fusion) for the 6-, 8-, and 10-min time points was calculated in each fusion reaction in the top panel, and the slope from linear curve fits was calculated for each reaction. The slopes were then averaged and compared with curve fits of the same time points taken from SNARE-only reactions. More dramatic increases in fold stimulation were observed with initial rates than endpoint fusion, as wild-type Munc18a produced a 4.48-fold increase in initial rate of fusion compared with a twofold increase in net fusion at 120 min. The impairment in fusion caused by the Munc18a E59R mutation was more noticeable in initial rate, as Munc18a E59R stimulated the initial rate of fusion by 2.83-fold, an 40% reduction from the initial rate of wild-type Munc18a. Little deviation in rate was observed with Munc18a R39E compared with wild-type (4.29- vs. 4.48-fold), whereas the initial rate of fusion of the phosphomimetic was 20% faster than wild-type (5.56- vs. 4.48-fold). Error bars, SEM.

Stimulation of Fusion by Munc18a Is Specific to Syntaxin1a/SNAP25b and VAMP2
The specificity of Munc18a for Syntaxin1a/SNAP25b and VAMP2 has been demonstrated by others with combinations of neuronal t-SNARE complexes and v-SNAREs from plasma membrane as well as other cellular compartments (Shen et al., 2007). Here, we tested the effects of Munc18a on the exocytic yeast t-SNAREs Sso1p/Sec9c and the cognate v-SNARE Snc1p. Addition of 20 µM Munc18a to fusion reactions of Sso1p/Sec9c and Snc1p failed to stimulate fusion to any degree (Figure 5B, bottom). Although Snc1p eliminates the stimulatory effect of Munc18a on Syntaxin1a/SNAP25 (Shen et al., 2007), it was unclear if Munc18a would affect fusion of Sso1p/Sec9c and VAMP2. This combination of yeast t-SNAREs and neuronal v-SNARE was fully fusogenic, and addition of 20 µM Munc18a produced a small but reproducible 1.2-fold stimulation of fusion (Figure 5B, top).

View larger version (26K): [in this window] [in a new window]   Figure 5. Stimulation by Munc18a is specific to neuronal SNAREs and requires the Syntaxin1a H3 domain but not SNAP25b. (A) Munc18a stimulates fusion of Syntaxin1a/Sec9c and VAMP2. Representative kinetic fusion graph of stimulation of Syntaxin1a/H8-Sec9c by Munc18a compared with fusion reactions containing equivalent amounts of Syntaxin1a/H6-SNAP25b. Difficulty in expression of Syntaxin1a/H8-Sec9c necessitated the use of liposomes with 1:500 protein:lipid ratios as determined by Amido Black protein quantitation and lipid recovery as measured by scintillation count of 3H in the lipid. Wild-type Syntaxin1a/H6-SNAP25b liposomes were reconstituted in matching protein:lipid ratios. Soluble wild-type Munc18a was added to a final concentration of 20 µM in both cases, and the final volume of all reactions was 60 µl. The same v-SNARE population (1:30 protein:lipid ratios) was used in each experiment. Each fusion reaction was conducted as described in Figure 1A. Background signal was measured by inhibiting fusion reactions with 20 µM CDV (solid line, top and bottom). Munc18a stimulates endpoint fusion of Syntaxin1a/H8-Sec9c (bottom, ) compared with SNARE-only reactions (bottom, ). Munc18a also stimulates equivalent reactions with wild-type Syntaxin1a/H6-SNAP25b (top, ) compared with SNARE-only reactions (top, ). However, the initial rate of fusion is significantly impaired when Munc18a is added to Syntaxin1a/H8-Sec9c compared with addition of Munc18a to Syntaxin1a/H6-SNAP25b as shown in Figure 7. (B) Stimulation of fusion by Munc18a is specific to neuronal SNAREs. Representative kinetic fusion graph of the effect of 20 µM Munc18a on fusion of the yeast t-SNAREs Sso1p/Sec9c and the neuronal v-SNARE VAMP2 (top) versus the yeast v-SNARE Snc1p (bottom). Data are reported as % maximum fluorescence. The fusion assay was performed as described in Figure 1A. Background signal was measured by inhibiting a fusion reaction with 20 µM CDV (solid line). An 1.2-fold stimulation of fusion resulted from addition of 20 µM Munc18a to fusion of Sso1p/Sec9c and VAMP2 (top, ) compared with SNARE-only Sso1p/Sec9c and VAMP2 (top, ). However, stimulation of fusion was completely abolished when 20 µM Munc18a was added to fusion of Sso1p/Sec9c and Snc1p (bottom, ) compared with SNARE-only Sso1p/Sec9c and Snc1p reactions (bottom, ). (C) The Syntaxin1a H3 domain is required for stimulation of fusion. Representative kinetic fusion graph of the effect of 20 µM Munc18a on fusion of the Syntaxin1a NRD/Sso1 H3 domain/SNAP25b chimera fused with VAMP2. Fusion reactions were conducted as described in Figure 1A, and data are reported as % of maximum fluorescence. Background signal was measured by inhibiting a fusion reaction with 20 µM CDV (solid line). Addition of 20 µM Munc18a had no observable effect on fusion of Syntaxin1a NRD/Sso1 H3 domain/SNAP25b and VAMP2 () compared with SNARE-only reactions ().

Replacement of the Syntaxin1a H3 Domain Abolishes Stimulation of Fusion
The results with the mixed t-SNARE complex suggest that interactions between Munc18a and the t-SNARE light chains of SNAP25 are minimal in Munc18a bound to the t-SNARE complex. However, the binary Munc18a/Syntaxin1a structure reveals extensive contacts between Munc18a and the H3 SNARE domain of Syntaxin1a. This interaction was further supported by recent calorimetric data from binding of soluble truncated Syntaxin1a mutants and Munc18a (Burkhardt et al., 2008). We next tested the functional consequences of replacing the H3 domain of Syntaxin1a with that of the yeast homolog Sso1p. A chimera comprised of the NRD of Syntaxin1a and the H3 domain of yeast Sso1p was paired with Sec9c as the t-SNARE light chain. This t-SNARE complex contains the neuronal Syntaxin1a NRD and three yeast SNARE domains in order to negate Munc18a interactions with the Syntaxin1a H3 domain and effectively isolate the Syntaxin1a NRD. This chimeric SNARE complex was fully functional, fused comparably to wild-type SNAREs or the mixed t-SNAREs (Figure 5C, ), and was inhibited fully by 20 µM CDV (solid line). However, no stimulation of the initial rate or final extent of fusion above control reactions was observed in the presence of 20 µM Munc18a (Figure 5C, , see also Figure 7).

Deletion of the Syntaxin1a Linker Region Abrogates Stimulation of Fusion
Syntaxin1a is known to assume either an "open" or a putatively autoinhibitory "closed" conformation (Dulubova et al., 1999). The Syntaxin1a NRD is thought to be able to change conformation via a flexible -helical linker region (residues 159-182; Margittai et al., 2003). Munc18a has been implicated in the regulation of the Syntaxin1a NRD conformational state (Dulubova et al., 1999; Yang et al., 2000). To determine if the Syntaxin1a linker region affects stimulation of fusion by Munc18a, a Syntaxin1a linker region deletion mutant (Syntaxin1a 159-182) was generated, coexpressed with SNAP25b, reconstituted, and fused with VAMP2 in the presence or absence of 20 µM Munc18a. Liposomes containing t-SNARE complexes produced with Syntaxin1a 159-182 fused normally with VAMP2 (Figure 6, ). However, stimulation of fusion in the presence of Munc18a was impaired markedly compared with wild-type Syntaxin1a/SNAP25 for this mutant (Figure 6, ). Both the rate (1.65- vs. 4.6-fold for wild type) and extent (1.3- vs. 2-fold for wild type) of fusion stimulation by Munc18a were largely abolished (Figure 7), whereas binding of Munc18a to free Syntaxin1a 159-182-H₆ was apparently unaffected (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 6. Deletion of the Syntaxin1a linker region largely abolishes stimulation of fusion. Kinetic fusion graph of Syntaxin1a159-182/H6-SNAP25b acceptor proteoliposomes fused with VAMP2 donor proteoliposomes in the presence () or absence of 20 µM Munc18a (). Fusion of Syntaxin1a159-182/H6-SNAP25b and VAMP2 without Munc18a was compared with equivalent wild-type Syntaxin1a/H6-SNAP25b and VAMP2 reactions (). The fusion assay was performed as described in Figure 1A, with final volumes of 60 µl/reaction. The same population of VAMP2 donor proteoliposomes was used in all reactions. Results are reported as % maximum fluorescence. Background signal was measured by inhibiting a fusion reaction with 20 µM CDV. Fusion of Syntaxin1a159-182/H6-SNAP25b was not impaired compared with wild-type Syntaxin1a/H6-SNAP25b. However, Munc18a was only able to stimulate fusion of Syntaxin1a159-182/H6-SNAP25b by 20%, very similar to the effect of Munc18a on Sso1p/Sec9c and VAMP2.

View larger version (17K): [in this window] [in a new window]   Figure 7. Summary of contribution of individual SNARE complex domains to Munc18a-mediated stimulation of fusion. Histogram of fold stimulation of net fusion at 120min (top) and of initial rate of fusion (6–10 min; bottom) by addition of 20 µM Munc18a to fusion reactions in which neuronal SNARE domains have been replaced with their homologous yeast SNARE domains. A maximum 2.2-fold stimulation in endpoint fusion was observed by addition of 20 µM Munc18a to fusion reactions of wild-type Syntaxin1a/SNAP25b and VAMP2, whereas the initial rate was stimulated 4.64-fold. Replacement of the t-SNARE light-chain SNAP25b with the yeast t-SNARE Sec9c resulted in an 20% decrease in net endpoint fusion, but entailed a dramatic 60% decrease in stimulation of initial rate from 4.64- to 1.96-fold. Deletion of the Syntaxin1a linker region (residues 159-182) resulted in an 40% reduction in stimulation of endpoint fusion (1.27-fold stimulation vs. 2.03-fold for wild-type), whereas stimulation of initial rate decreased by 65% (1.65- vs. 4.64-fold for wild-type). Replacement of the SNARE domains of the t-SNARE complex light chain with equivalent helices from Sec9c combined with replacement of the Syntaxin1a SNARE (H3) domain with the Sso1 SNARE domain, leaving the Syntaxin1a NRD as the only native piece of the Syntaxin1a/SNAP25b t-SNARE complex resulted in essential abrogation of stimulation of endpoint fusion as well as initial rate. Surprisingly, a 1.3-fold stimulation of endpoint fusion of Sso1/Sec9c and VAMP2 was observed, with an equivalent increase in initial rate. Munc18a was without effect on endpoint fusion or initial rate of Sso1p/Sec9c and Snc1p.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Among all the SM proteins, Munc18a is of special interest as it specifically regulates neurotransmitter release. The atomic structure of a binary Munc18a/Syntaxin1a complex showed extensive interactions between the Syntaxin1a NRD (Habc domain) and H3 core domains with Munc18a (Misura et al., 2000). Recent refinement of this crystal structure revealed an additional interaction between the extreme N-terminus of Syntaxin1a with Munc18a (Burkhardt et al., 2008), which has also been recently detected by NMR (Khvotchev et al., 2007). SM protein interaction with extreme N-terminal sequences has also been demonstrated for Munc18c (Latham et al., 2006), and the ER SM protein Sly1p (Bracher and Weissenhorn, 2002; Yamaguchi et al., 2002). This mode of interaction has important implications for previous binding studies that utilized N-terminally tagged Syntaxin1a (Rodkey and McNew, 2008). These interactions apparently stabilize Syntaxin1a in its closed conformation and prevent the formation of the t-SNARE complex, which is consistent with the negative role of Munc18a.

Recent studies have shown that Munc18a could specifically stimulate SNARE-mediated in vitro membrane fusion with neuronal SNAREs (Shen et al., 2007), which extended our previous observation that yeast Sec1p stimulates membrane fusion in vitro (Scott et al., 2004). Although a larger magnitude of stimulation was observed in this recent study (Shen et al., 2007), the degree of stimulation by Munc18a can be largely attributed to differences in the concentration of SNAREs in the proteoliposomes. We confirm and extend these results by showing that Munc18a stimulates fusion by neuronal SNAREs (Figure 1). Furthermore, we see that the rate enhancement that Munc18a provides is limited to early time points in the reaction and is largely complete within the first 20 min (Supplemental Figure S1), an effect similar to that seen previously for Munc18a (Shen et al., 2007). The underlying basis for this time-limited effect remains to be determined. We also find that fusion stimulation by Munc18a requires that all SNARE participants be present during preincubation of the reaction (Figure 2) and define Munc18a bound to the t-SNARE complex as a functional intermediate (Figure 3).

Given that the binary Munc18a/Syntaxin1a complex appears incompatible with further SNARE complex formation, we probed the associations required for Munc18a interactions with the assembled t-SNARE complex. The main goal was to determine if molecular interactions important for the binary Munc18a/Syntaxin1a were preserved when SNAP25 was present. We approached this task by mutational analysis of key residues within Munc18a (Figure 4) observed in the Munc18a/Syntaxin1a crystal structure as well as by interchanging SNARE domains with the homologous yeast SNARE complex (Figure 5).

We examined the effect of disrupting ionic interactions between the NRD or the H3 core domain of Syntaxin1a and Munc18a (Figure 4). We found that mutation of Munc18a E59 to arginine, which is predicted to disrupt an ionic interaction in Helix C (Syn1a R114-Munc18a E59) within the NRD, was much more detrimental to the ability of Munc18a to stimulate fusion than a similar charge reversal (R39E) that interacts with a central region of the H3 domain (Syn1a E234). These results suggest that the R114-E59 interaction is maintained and functionally relevant in the ternary complex between Syntaxin1a/SNAP25 and Munc18a, whereas the E234-R39 interaction is less important for t-SNARE complex–bound Munc18a. Consistent with our results, mutation of Munc18a R39 to Cys had little or no impact on stimulation of fusion (Shen et al., 2007).

Next, we addressed the effects of individual SNARE domains by changing the composition of the assembled t-SNARE complex. Munc18a fails to stimulate fusion with plasma membrane yeast SNAREs (Figure 5B), which suggests that systematic replacement of neuronal t-SNARE complex domains with their yeast homologues would effectively negate interaction of Munc18a with the replaced domains. We began by expressing a novel "mixed t-SNARE complex" composed of Syntaxin1a and Sec9c designed to analyze the contribution of the SNAP25 SNARE chains. Not only was this hybrid t-SNARE complex functional, it was stimulated by Munc18a to a similar extent as the fully neuronal SNARE complex (Figure 5A). This result indicates that Munc18a does not associate significantly with SNAP25b in the assembled t-SNARE complex and highlights the importance of Munc18a/Syntaxin1a interactions even within the assembled t-SNARE complex.

Because replacing SNAP25 with Sec9c had only minor effects, we modified the t-SNARE complex further by substituting the Syntaxin1a H3 domain within the mixed t-SNARE complex with the corresponding yeast Sso1p H3 domain, leaving the Syntaxin1a NRD as the sole neuronal piece of the t-SNARE complex (Figure 5C). When this Syntaxin1a NRD/Sso1p H3 chimera was paired with Sec9c, stimulation of fusion by Munc18a was abolished entirely, indicating that the Syntaxin1a H3 domain is a crucial determinant of the ability of Munc18a to stimulate fusion. Although the interaction of the Syntaxin1a H3 domain and Munc18a is required for in vitro fusion stimulation, it is unlikely that the configuration of the H3 domain seen in the crystal structure is maintained in the Munc18a bound t-SNARE complex considering the small detrimental effect caused by the Munc18a R39E mutant.

Overall, the results of mutations in Munc18a and systematic domain replacements within the t-SNARE complex indicate that Munc18a binds the assembled t-SNARE complex in a fundamentally different manner than free Syntaxin1a, although some of the same contacts found in the binary Munc18a/Syntaxin1a complex may be retained.

Our results, paired with the recent appreciation that the extreme N-terminus of Syntaxin1a also interacts with Munc18a, suggests a possible path describing the transition from the binary Munc18a/Syntaxin1a complex to Munc18a bound t-SNARE complex through a Munc18a bound trans-SNARE complex. The ability of Munc18a to stimulate fusion in vitro absolutely requires an intact Syntaxin1a N-terminus. Stimulation is lost when the entire NRD is removed (data not shown), when the N-terminal 19 amino acids are truncated and even when a single point mutation (L8A) is introduced (Shen et al., 2007). We suggest that this point of interaction is critical for the binding of Munc18a to the assembled t-SNARE complex and likely part of the mechanism that allows t-SNARE complex formation to occur while Syntaxin1a remains bound to Munc18a. We found that the H3 core domain of Syntaxin1a was absolutely required for Munc18a stimulation, yet the SNARE domains of SNAP25 have minimal effect on the ability of Munc18a to stimulate fusion (Figure 5). We also determined that the linkage between the NRD and the H3 core helix is critically important for Munc18a stimulation (Figure 6). These data fit a model in which movement of the Syntaxin1a NRD away from the H3 core domain within the binary Munc18a/Syntaxin1a complex allows SNAP25 binding, but requires an extreme N-terminal attachment to maintain contact with Munc18a during and after this transition. This hypothetical movement of the NRD would also require that the linkage between the NRD and the H3 core helix be flexible enough to permit this transition. Removal of this flexible linker region should prevent the NRD from being moved or stretched by Munc18a, preventing productive association.

Although the Munc18a/t-SNARE complex interaction clearly is necessary for stimulation of fusion, order of addition experiments indicate that accelerated fusion cannot occur unless all components, including the v-SNARE, are incubated together (Figure 2). A precedent for SM/v-SNARE interaction was established by work on Vps45 and Snc1p (Carpp et al., 2006). Although we were able to detect weak Munc18a/VAMP2 association biochemically, a functional Munc18a/VAMP2 interaction is suggested by our in vitro fusion results. Munc18a has absolutely no effect on the full yeast SNARE complex (Sso1p/Sec9c;Snc1p Figure 5B, bottom) but shows a reproducible 30% stimulation with the yeast t-SNARE complex (Sso1p/Sec9c) and VAMP2. The most straightforward interpretation of these data are that VAMP2 interacts with Munc18a, whereas Snc1p does not. This interpretation is bolstered by mutations in VAMP2 that diminished the ability of Munc18a to stimulate fusion (Shen et al., 2007).

The end result of these proposed structural changes would be that Munc18a binding actively positions the Syntaxin1a NRD in a conformation favorable to fusion, first for t-SNARE complex formation and ultimately for trans-SNARE complex formation. Hypothetical models indicate that Munc18a could effectively "roll" the Syntaxin1a NRD so that it allows SNAP25 binding and exposes the VAMP2-binding groove in the t-SNARE complex, thereby facilitating VAMP2 binding and consequent membrane fusion (McNew, 2008).

Taken together, these results place Munc18a as an assembly platform that may orchestrate t-SNARE complex formation and trans-SNARE complex assembly that stimulates membrane fusion by placing the Syntaxin1a NRD in a position that facilitates t-SNARE complex/v-SNARE interaction. In addition, Munc18a may actively recruit and position VAMP2 to facilitate fusion. These in vitro fusion results support the idea that Munc18a facilitates post-Golgi vesicle fusion with the plasma membrane in neurons.

	ACKNOWLEDGMENTS

 
The authors thank Drs. Kirilee Wilson and Brenton Scott for preliminary observations and Dr. Blair Doneske, Joseph Faust, and Tyler Moss (All three from Rice University) for kind gifts of protein and liposomes, as well as for advice. The authors also thank other members of the McNew Lab for close reading of the manuscript. Support for this work was provided by National Institutes of Health Grant GM71832 and the G. Harold and Leila Mathers Charitable Foundation.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-05-0538) on October 1, 2008.

Address correspondence to: James A. McNew (mcnew{at}rice.edu).

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