Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Aug 3.
Published in final edited form as: Cell Metab. 2011 Aug 3;14(2):254–263. doi: 10.1016/j.cmet.2011.07.002

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis

Chuenchanok Khodthong 1, Greg Kabachinski 1, Declan J James 1, Thomas FJ Martin 1
PMCID: PMC3148490  NIHMSID: NIHMS311491  PMID: 21803295

Abstract

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca2+-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca2+-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identified a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca2+-triggered exocytosis of either docked or newly-arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.

INTRODUCTION

The release of neuropeptides and peptide hormones in neurons and endocrine cells by Ca2+-dependent vesicle exocytosis is a tightly controlled process catalyzed by SNARE protein complexes (Jahn and Scheller, 2006). Vesicle exocytosis employs the v-SNARE protein VAMP-2 (aka synaptobrevin) on the vesicle that assembles with the plasma membrane t-SNARE proteins syntaxin-1 and SNAP-25 into four-helix bundle complexes that bridge membranes to promote bilayer fusion (Sutton et al., 1998; Weber et al., 1998). To account for the rapid fusion of dense-core vesicles following Ca2+ influx in chromaffin cells, it was proposed that SNARE complexes are assembled in advance on docked vesicles during a priming step (Rettig and Neher, 2002). Undocked vesicles in endocrine cells also undergo rapid evoked exocytosis, which indicates that priming can also occur immediately upon vesicle arrival at the plasma membrane. Indeed, studies in pancreatic β cells detected preassembled SNARE complexes at sites of rapid exocytosis as well as SNARE complex assembly immediately preceding exocytosis that followed Ca2+ influx (Takahashi et al., 2010). The molecular basis for vesicle priming and SNARE complex assembly is poorly understood.

Genetic and biochemical studies indicate that CAPS (Ca2+-dependent activator protein for secretion) and related Munc13 proteins prime vesicle exocytosis (Stevens and Rettig, 2009; Wojcik and Brose, 2007). CAPS (aka CADPS, UNC31) is a neural/endocrine-specific protein that reconstitutes Ca2+-triggered dense-core vesicle exocytosis in permeable neuroendocrine cells (Ann et al., 1997; Walent et al., 1992) and in purified plasma membranes with docked dense-core vesicles (Martin and Kowalchyk, 1997), which indicates function after vesicle docking. In accord with this, neuroendocrine cells lacking CAPS exhibit impaired vesicle exocytosis without impaired vesicle docking (Fujita et al., 2007; Grishanin et al., 2004; Liu et al., 2008; Nojiri et al., 2009). In chromaffin and pancreatic β cells, CAPS was shown to function at a vesicle priming step (Elhamdani et al., 1999; Liu et al., 2008; Olsen et al., 2003; Speidel et al., 2008). Reductions in docked dense-core vesicles in pancreatic β cells or C. elegans neurons lacking CAPS may also indicate a role in stabilizing vesicle docking (Hammarlund et al., 2008; Speidel et al., 2008). The mechanism that CAPS utilizes at a late step in dense-core vesicle exocytosis is not fully understood.

CAPS contains several possible functional domains (Fig. 1A). A central pleckstrin homology (PH) domain binds PI(4,5)P2 for plasma membrane recruitment (Grishanin et al., 2002; Grishanin et al., 2004). A region in the C-terminal half of CAPS, which exhibits homology (20% identity, 40% similarity) to a region in the C-terminal half of Munc13-1, contains two contiguous annotated homology domains, DUF1041 (domain of unknown function 1041; pfam 06292) and MHD1 (Munc13 homology domain-1) (Koch et al., 2000), referred to as the DAMH (DUF1041 and MHD1) domain. The DAMH domain is conserved in all CAPS/Munc13 family proteins but its functional role is unknown.

Figure 1. SNARE binding in CAPS localizes to DAMH domain.

Figure 1

Open in a new tab

A. Schematic of CAPS GST fusion proteins. Black boxes indicate C2, PH and DAMH domains. Grey box indicates homology to Munc13. B. GST fusion proteins at 0.2 μM were assayed for binding to 0.5 μM t-SNAREs in PC liposomes. Liposome-bound proteins were isolated from gradients and analyzed on SDS-PAGE for Western blotting with GST antibody (top panel) or syntaxin-1 antibody (bottom panel). CAPS(859-1073) corresponds to DAMH protein. C. Binding to t-SNAREs in PC liposomes was conducted at the indicated concentrations of MBP-DAMH. Liposome-bound MBP-DAMH was analyzed by SDS-PAGE with SYPRO Ruby staining and densitometry. Best fit curve indicated binding parameters: Kd = 177 ± 47 nM and Bmax=0.3 mol CAPS bound/mol t-SNAREs (mean ± SE, n=3). D. Competition for binding of 0.3 μM GST-DAMH to 0.5 μM t-SNAREs in PC liposomes was conducted for indicated CAPS concentrations. Liposome-bound proteins were analyzed by Western blotting with CAPS antibody (top and middle panels) or syntaxin-1 antibody (bottom panel) and by densitometry. Competition by 2 μM CAPS in duplicate with ranges is indicated. E. 0.5 μM t-SNAREs, syntaxin-1 or SNAP-25 in liposomes and their protein-free counterparts were incubated with 0.5 μM GST-DAMH. Bound GST-DAMH was analyzed by SDS-PAGE and SYPRO Ruby staining. SNARE binding by GST-DAMH, normalized to the SNARE content of the liposomes, was expressed as mean values ± SE (n=6). F. 0.3 μM GST-DAMH was incubated with 0.5 μM t-SNAREs or thrombin-cleaved t-SNAREs in PC liposomes and the bound fraction was analyzed by Western blotting with CAPS antibody (top panel), syntaxin-1 antibody (middle panel) or SNAP-25 antibody (bottom panel). SNARE binding by GST-DAMH, normalized to the SNARE content of the liposomes, was expressed as mean values ± SE (n=7). Diagram of the domain organization of syntaxin-1 with thrombin cleavage site indicated by arrow.

Recent studies showed that CAPS stimulates trans SNARE complex formation on liposomes and is able to promote the SNARE-dependent fusion of liposomes that contain PI(4,5)P2 (James et al., 2008; James et al., 2009). CAPS was also found to bind to each of the three exocytic SNARE proteins, syntaxin-1, SNAP-25, and VAMP-2 (Daily et al., 2010). These findings suggest possible mechanisms for CAPS in driving SNARE complex assembly during priming. However, the domain in CAPS responsible for SNARE binding and the relevance of SNARE binding to CAPS function in cells have not been determined. Here we identify a SNARE-binding region in CAPS within the DAMH domain corresponding to MHD1 and show that it is essential for CAPS activity at a late step in dense-core vesicle exocytosis.

RESULTS

The SNARE-binding region in CAPS corresponds to the DAMH domain

CAPS interacts with membrane-integrated heterodimeric t-SNARE complexes comprised of syntaxin-1 and SNAP-25 (Daily et al., 2010). To identify the t-SNARE-binding domain in CAPS, fusion proteins across the full length of CAPS were generated (Fig. 1A). These were designed to correspond to folded domains in the protein based on fragments generated by limited protease digestion (Loyet et al., 1998). The GST-fusion proteins, each at 0.2 μM, were incubated with t-SNARE-containing liposomes and bound proteins were separated from unbound by buoyant density gradient centrifugation. The fusion protein corresponding to the DAMH domain (CAPS 859-1073) efficiently bound to membrane-integrated t-SNAREs whereas other fusion proteins did not (Fig. 1B). Under these conditions, the binding of fusion proteins to protein-free liposomes was negligible (not shown).

Binding studies over a range of DAMH domain protein concentrations at a constant concentration of t-SNARE-containing liposomes revealed a dissociation constant Kd = 177 ± 47 nM (Fig. 1C), which is similar to that for full-length CAPS binding to t-SNARE proteins (Daily et al., 2010). Full-length CAPS effectively and fully competed with the DAMH domain protein for binding to t-SNAREs (Fig. 1D). Conversely, t-SNARE binding by full-length CAPS was effectively competed by the DAMH domain protein (Fig. S1A). The identification of the DAMH domain protein as the only t-SNARE-binding fragment in CAPS and its ability to fully compete for CAPS binding indicate that the DAMH domain is the major site in CAPS for t-SNARE binding.

The DAMH domain interacts with individual t-SNARE proteins

We characterized the t-SNARE-binding properties of the DAMH domain protein to compare with those of the full-length CAPS protein. The DAMH domain protein interacted with the individual membrane-associated t-SNARE proteins, syntaxin-1 and SNAP-25, to a similar extent and exhibited two-fold greater binding to membrane-integrated heterodimeric t-SNARE complexes (Fig. 1E). This indicated that syntaxin-1 and SNAP-25 each contribute a site for DAMH domain protein binding in heterodimeric t-SNARE complexes. Syntaxin-1 contains several domains (Fig. 1F, lower) including an N-terminal Habc domain, a membrane proximal helical H3 domain (SNARE motif), and a C-terminal transmembrane domain. To determine the binding site for the DAMH domain protein on syntaxin-1, we introduced a thrombin cleavage site between the Habc and H3 domains (Parlati et al., 1999). DAMH domain protein binding to heterodimeric t-SNARE complexes was similar with or without thrombin cleavage (Fig. 1F), showing that the binding site for the DAMH domain protein is in the C-terminal H3-linker region of syntaxin-1. Thus, the DAMH domain protein appeared to exhibit the same novel characteristics of full-length CAPS in binding to individual t-SNARE proteins and to the H3-linker region in syntaxin-1 (Daily et al., 2010).

Competition binding studies confirmed the preferential binding of the DAMH domain protein to C-terminal but not N-terminal regions of syntaxin-1 (Fig. S1B). Moreover, these competition studies revealed that the DAMH domain protein contains a single site for binding t-SNARE proteins or two distinct sites (one for syntaxin-1 and one for SNAP-25) that overlap.

The DAMH domain interacts with VAMP-2

Full-length CAPS interacts with each of the exocytic SNARE proteins including VAMP-2 (Daily et al., 2010) but the binding site in CAPS for VAMP-2 has not been identified. We found that the DAMH domain protein bound to membrane-integrated VAMP-2 with a Kd = 212 ± 73 nM (Fig. 2A). The low stoichiometry of binding may be due to the limited folding of VAMP-2 in isolation on membranes (Ellena et al., 2009). The DAMH domain is likely the main VAMP-2 binding site in full-length CAPS because the DAMH domain protein effectively competed with full-length CAPS for binding to membrane-integrated VAMP-2 (Fig. S1C). The results indicate that the DAMH domain protein interacts with each of the exocytic SNARE proteins as does full-length CAPS.

Figure 2. Localization of SNARE binding to MHD1.

Figure 2

Open in a new tab

A. Indicated concentrations of MBP-DAMH were incubated with 0.5 μM VAMP-2 in PC liposomes and liposome-bound protein was quantified by SYPRO Ruby staining (mean ± SE, n=3). A best fit curve for one binding site indicated Kd = 212 ± 73 nM and Bmax = 0.09 mol bound DAMH/mol VAMP-2. B. t-SNAREs, ternary complex (t-SNAREs + VAMP-2(1-94)) or ternary complex with bound complexin-I at 0.5 μM in PC liposomes were incubated with 0.3 μM GST-DAMH. Liposome-bound GST-DAMH was analyzed by SDS-PAGE with SYPRO Ruby staining. Relative GST-DAMH binding is shown as mean ± SE (n=6). C. Schematic of the MBP-CAPS fusion proteins within DAMH domain with predicted secondary structure indicated. Panel on the right summarizes binding to t-SNARE- and v-SNARE-containing liposomes for each protein. D. MBP-CAPS proteins at 0.2 μM were incubated with 0.5 μM t-SNAREs in PC liposomes and gradient fractions were analyzed by Western blotting with MBP antibody. Liposome-bound protein was in fraction 2. Similar studies were conducted with v-SNARE liposomes. E. Schematic of similarity for helical segments in MHD1 (CAPS957-983) and VAMP-2(31-56) from ClustalW alignment (*-indicates identity, :/.-indicates strongly/weakly similar). Amino acids in VAMP-2 in interacting layers of SNARE complexes are boxed. F. Schematic of CAPS wild-type and CAPS(Δ952-997) mutant. G. Full-length CAPS and CAPS(Δ952-997) at 0.2 μM were incubated with 0.5 μM t-SNAREs in PC liposomes. Bound protein (fractions 1 and 2) was separated from free (fractions 5 and 6) by gradient flotation and analyzed by immunoblotting with CAPS or syntaxin-1 antibody.

We found that the DAMH domain protein also interacted with ternary SNARE complexes that were formed by binding soluble VAMP-2 to t-SNARE liposomes. DAMH domain protein binding to ternary SNARE complexes (with or without bound complexin-1) was similar to binding to t-SNARE complexes (Fig. 2B). This indicated that the binding sites for the DAMH domain protein on syntaxin-1 H3 and SNAP-25 remain accessible after VAMP-2 is bound. It was noteworthy that VAMP-2 incorporation into t-SNARE complexes to form ternary SNARE complexes did not contribute an additional binding site for the DAMH domain protein (Fig. 2B). This likely indicates that the binding site for the DAMH domain on VAMP-2 is masked when VAMP-2 is bound into the ternary SNARE complex. These results may indicate that CAPS interacts with VAMP-2 prior to but not after the formation of a ternary SNARE complex.

The preceding studies revealed that the 215 residue DAMH domain protein recapitulates many of the SNARE-binding properties of the full-length 1289 residue CAPS protein. In competition binding studies (Fig. S1D), we found that v-SNARE and t-SNARE binding by the DAMH domain protein was likely mediated through distinct but overlapping sites.

A helix within MHD1 mediates SNARE binding

The DAMH domain is predicted to be largely helical with 4-5 α helices joined by short unstructured segments. To identify a core SNARE-binding domain in CAPS, we generated fusion proteins that span one or several of the predicted helices for testing t-SNARE and v-SNARE binding (Fig. 2C and D). A fusion protein corresponding to part of MHD1 (residues 929–997) was found to interact with t-SNARE- or with v-SNARE-containing liposomes. Thus, a core SNARE-binding domain appeared to consist of a 69 residue portion of MHD1. The larger of the predicted helices in this region (box in Fig. 2C) contained sequences that exhibit homology (67% similar) to the N-terminal half of the SNARE motif in VAMP-2 (Fig. 2E).

This predicted helix in the MHD1 region encompassed residues 952-997. A full-length CAPS protein lacking this region (Fig. 2F) was expressed, purified, and tested for its ability to interact with membrane-integrated t-SNAREs by buoyant density flotation (Fig. 2G). Whereas a significant fraction of wild-type CAPS protein bound to t-SNARE-containing liposomes, there was negligible binding of the CAPS(Δ952-997) protein to t-SNARE-containing liposomes (Fig. 2G). The CAPS(Δ952-997) protein retained other properties of the wild-type protein such as lipid binding and mobility on native gels (not shown) suggesting that it was properly folded. The results indicate that t-SNARE binding by CAPS requires a helix within MHD1.

Functional analysis of SNARE-binding-deficient CAPS mutant

The function of CAPS can be assessed by its activity in reconstituting Ca2+-dependent vesicle exocytosis in permeable neuroendocrine cells (Grishanin et al., 2004). Whereas 20 nM wild-type CAPS effectively reconstituted Ca2+-triggered vesicle exocytosis in permeable PC12 cells, the activity of 40 nM CAPS(Δ952-997) was greatly reduced (Fig. 3A). When tested together, the CAPS(Δ952-997) protein significantly reduced the activity of the wild-type CAPS protein, which indicates that the mutant protein exhibits dominant inhibitory properties. Overall, the CAPS(Δ952-997) protein exhibited a strong loss-of-function for reconstituting Ca2+-dependent vesicle exocytosis in permeable cells that was comparable to its strong loss of SNARE binding.

Figure 3. Properties of CAPS(Δ952-997) mutant.

Figure 3

Open in a new tab

A. Testing CAPS(Δ952-997) for reconstitution of Ca2+-triggered vesicle exocytosis in permeable PC12 cells. Permeable cells were incubated at 35°C for the indicated times with 20 nM CAPS, 40 nM CAPS(Δ952-997) or both after injection of 1 μM Ca2+ at zero time. The rotating disk electrode detected norepinephrine as a voltage. B. Testing a DAMH domain protein for reconstitution of Ca2+-triggered vesicle exocytosis in permeable PC12 cells. Studies were conducted as in A with 10 nM CAPS, 30 μM DAMH protein or CAPS with varying DAMH protein concentrations as indicated. C. PC12 cells expressing CAPS-mKate2 or CAPS(Δ952-997)-mKate2 as indicated with syntaxin-1-EGFP were imaged by two channel TIRF microscopy (left panels). Representative line scans (right panels) between the arrowheads indicated on the left panels were taken after thresholding images to cover multiple syntaxin-1 clusters. These indicated that CAPS-mKate2 clusters colocalized with syntaxin-1-EGFP clusters (upper right panel) whereas CAPS(Δ952-997)-mKate2 failed to localize to the plasma membrane with syntaxin-1-EGFP clusters (lower right panel). Colocalization was quantified in several (n=12) images similar to those in the left panels as the thresholded Mander’s split colocalization coefficient where 1.0 is full colocalization and 0.0 is no colocalization. Syntaxin-1-EGFP clusters colocalized with CAPS-mKate2 puncta with a coefficient = 0.36 ± 0.08. This was substantially reduced (to 0.02 ± 0.01) for syntaxin-1-EGFP colocalization with CAPS(Δ952-997)-mKate2.

These data indicated that sequences in MHD1 are necessary for CAPS activity. Because the DAMH domain protein recapitulated many of the SNARE-binding properties of CAPS, we also determined whether the DAMH domain might be sufficient for CAPS function. However, we failed to detect a stimulatory effect of the DAMH domain protein in the permeable cell assay (Fig. 3B). By contrast, increasing concentrations of the DAMH domain protein significantly inhibited CAPS stimulation of Ca2+-triggered catecholamine release. These studies show that sequences within the DAMH domain are necessary but not sufficient for CAPS activity.

We further tested the activity of the CAPS(Δ952-997) protein to support evoked vesicle exocytosis in live PC12 cells. We first determined the localization of CAPS(Δ952-997) compared to wild-type CAPS in PC12 cells by TIRF microscopy (Fig. 3C). Wild-type CAPS distributes as clusters that colocalize with clusters of syntaxin-1 (Fig. 3C, upper). By contrast, CAPS(Δ952-997) was diffusely distributed and failed to localize in clusters while syntaxin-1 clusters exhibited a normal distribution (Fig. 3C, lower). These results were consistent with a loss of SNARE binding for the CAPS(Δ952-997) protein.

To test the activity of CAPS proteins in evoked vesicle exocytosis, we expressed brain-derived neurotrophic factor (BDNF)-GFP to directly image plasma membrane-proximal dense-core vesicles by TIRF microscopy (Fig. 4A). Exocytosis of single vesicles was detected as increased BDNF-GFP fluorescence due to neutralization of an acidic vesicle upon fusion pore formation (Fig. 4C). Depolarization of PC12 cells stimulates Ca2+ influx, which promotes a rapid increase in the number of vesicle exocytic events (Fig. 4D). The shRNA-mediated down regulation of CAPS to 10% of control levels (Fig. 4G) strongly reduced the number of evoked exocytic events (Fig. 4D). Rescue studies were conducted with plasmids encoding shRNA-resistant CAPS-tagRFP proteins to simultaneously monitor protein expression and function. Re-expression of wild-type CAPS restored evoked exocytosis close to levels of wild-type cells (Fig. 4E). By contrast, expression of CAPS(Δ952-997) at levels similar to those of wild-type CAPS (Fig. 4H) failed to restore evoked exocytosis (Fig. 4E).

Figure 4. SNARE binding is essential for CAPS function.

Figure 4

Open in a new tab

PC12 cells were transfected with a plasmid encoding BDNF-GFP and CAPS-1 was down-regulated by expression of an shRNA plasmid. Rescue constructs were used to re-express wild-type CAPS or CAPS(Δ952-997). A. TIRF view of BDNF-GFP-containing dense-core vesicles in cells rescued with wild-type CAPS or CAPS(Δ952-997). B. Number of vesicles in the evanescent field for cells in panel A plotted as density (from 5 cells). Identical vesicle densities were found in wild-type and CAPS knockdown cells (not shown). C. 56 mM K+-containing buffer stimulates Ca2+ influx and promotes vesicle exocytosis detected as increased fluorescence of BDNF-GFP. Compiled profiles for single events (with maximal fluorescence aligned at t=0) is shown for rescue with wild-type CAPS (n=150) and CAPS(Δ952-997) (n=25). No significant differences were observed. D. and G. CAPS down regulation by shRNA decreased CAPS levels by 90% (G) and resulted in a 5-fold reduction in the number of exocytic events evoked by 56 mM K+ depolarization applied at zero time (D). Recordings were at 4Hz and events binned at each 15s are shown (means ± SE, n=11 cells). E. and H. Transfection with a pcDNA3.1-CAPS-tagRFP harboring silent mutations enabled equivalent re-expression of wild-type CAPS or CAPS(Δ952-997) in down-regulated PC12 cells (H). Wild-type CAPS expression restored evoked exocytosis whereas CAPS(Δ952-997) expression failed to do so (E) (means ± SE, n=12-13 cells). F. Analysis of fusion events from resident and non-resident vesicles. Lower panel shows representative fusion events recorded at 4 Hz from resident or non-resident vesicles defined by whether vesicle fluorescence was evident within 0.5s of fusion. The proportion of resident or non-resident fusion events (means ± SE, n=12-13 cells) is shown for CAPS knockdown, wild-type CAPS rescue or CAPS(Δ952-997) re-expression and was similar for wild-type cells (not shown).

Wild-type and CAPS knockdown cells (not shown) as well as cells re-expressing wild-type CAPS or the CAPS(Δ952-997) protein had similar numbers of membrane-proximal dense-core vesicles (Fig. 4A and B), which indicated that the loss of function occurred at a step following vesicle delivery to the plasma membrane. Most (~75%) exocytic events elicited by depolarization in PC12 cells arise from membrane-proximal vesicles (termed residents) but there are also some (~25%) exocytic events from vesicles that approach the plasma membrane during stimulation (termed non-residents) (Fig. 4F). Resident and non-resident exocytic events exhibit similar time courses following stimulation (Fig. S2). In spite of large differences in the total number of vesicle exocytic events in cells depleted for CAPS or rescued with wild-type CAPS or CAPS(Δ952-997) (Fig. 4D and E), there was no significant difference in the proportion of resident and non-resident vesicle fusion events (Fig. 4F). This indicates that CAPS is required for the exocytosis of both resident as well as newly-arrived non-resident vesicles, which suggests a function for CAPS after vesicle arrival at the plasma membrane. These results contrast with a reported preferential inhibitory effect of overexpressed Doc2α mutants on resident but not non-resident vesicle exocytosis (Sato et al., 2010). The kinetics of individual fusion events did not differ for CAPS knockdown cells rescued by wild-type CAPS or CAPS(Δ952-997) (Fig. 4C). This suggests that CAPS participates in determining vesicle fusion probabilities but is not directly involved in the fusion process. Our results indicate that CAPS lacking part of the SNARE-binding MHD1 domain fails to support evoked vesicle exocytosis, which implies that SNARE binding is essential for CAPS function.

DISCUSSION

SNARE proteins catalyze membrane fusion as four-helix bundle complexes that bridge vesicle and plasma membranes. The timing of SNARE complex assembly relative to evoked vesicle exocytosis has been assessed in pancreatic β cells (Takahashi et al., 2010) but the mechanisms that initiate SNARE complex assembly prior to exocytosis remain to be clarified. CAPS binds to SNARE proteins (Daily et al., 2010) and promotes trans SNARE complex formation on liposomes (James et al., 2009), which suggests that CAPS could function to assemble SNARE complexes in advance of or at the time of fusion. The current work showed that CAPS binding to SNARE proteins is essential for evoked vesicle exocytosis. Future studies will need to directly assess the role of CAPS in the assembly of SNARE complexes in neuroendocrine cells.

The principal SNARE-binding domain in CAPS was identified as corresponding to the DAMH domain, which consists of two adjacent (DUF1041 and MHD1) homology domains of unknown function. The SNARE-binding characteristics of a DAMH domain protein were remarkably similar to those of full-length CAPS, which include interactions with each of the exocytic SNARE proteins and the novel property of binding the C-terminal SNARE motif of syntaxin-1. A core SNARE-binding region in the DAMH domain appeared to correspond to MHD1. The lack of t-SNARE binding by the CAPSΔ952-997 mutant, which lacks a predicted helix within MHD1, is consistent with this assignment. The loss-of-function of the CAPSΔ952-997 protein in cells was accompanied by normal delivery of vesicles to the plasma membrane, similar losses in resident and non-resident vesicle fusion events, and a marked reduction in the number of vesicle fusion events without a change in their kinetics. The results indicate that SNARE binding by MHD1 is essential for CAPS function at a step following vesicle delivery to the plasma membrane.

This work provides the first identification of MHD1 as a SNARE-binding domain. A DAMH domain with MHD1 is present in all members of the CAPS/Munc13 family of proteins but whether MHD1 mediates SNARE binding by Munc13 proteins is currently uncertain. The interaction of a C-terminal region of Munc13-1 with an N-terminal domain of syntaxin-1 was reported in yeast two hybrid studies (Betz et al., 1997) but biochemical studies were unable to confirm this (Basu et al., 2005; Ma et al., 2011). Several mutations in Munc13-1/UNC13 including in MHD1 were identified that decreased binding to N-terminal syntaxin fragments (Madison et al., 2005; Stevens et al., 2005). However, recent studies suggested that the C-terminus of Munc13-1 may actually interact with the H3 SNARE domain of syntaxin (Ma et al., 2011) as does CAPS (Daily et al., 2010). Studies are needed to determine whether the DAMH domain and MHD1 mediate SNARE binding in Munc13 proteins. The main mechanism proposed for Munc13-1 is binding to syntaxin-1 to enable a transition from a Munc18-1-bound closed conformation to an open conformation that allows SNARE complex assembly (Ma et al., 2011; Wojcik and Brose, 2007). It is currently unclear whether CAPS functions similarly. Munc18-1 enhances CAPS binding to syntaxin-1 which suggests there may be functional interactions (Daily et al., 2010).

Full-length CAPS binds helical SNARE motifs of syntaxin-1, SNAP-25 and VAMP-2 but it exhibits specificity in binding to only a subset of exocytic SNARE protein isoforms (Daily et al., 2010). The current work showed that the DAMH domain protein interacts with the SNARE plus linker motif of syntaxin-1 as well as with SNAP-25 and VAMP-2. Competition binding studies suggested that the DAMH domain contains distinct but overlapping binding sites for v- and t-SNAREs. This region included sequences with homology to the N-terminal half of the SNARE motif in VAMP-2, which implies that part of the SNARE-binding region in CAPS has SNARE motif-like characteristics. However, the role of this VAMP-like region in CAPS is uncertain. It is unlikely that the VAMP-like motif in CAPS binds t-SNAREs in a manner similar to VAMP-2 because VAMP-2 pre-bound to t-SNAREs did not prevent DAMH domain protein binding. The VAMP-like segment in CAPS is part of a larger helical region and it could play a role as a coiled-coil trigger site as was suggested for cognate sequences of VAMP-2 (Wiederhold et al., 2010).

There is precedent for a SNARE motif-like domain residing in SNARE regulatory proteins. In the tethering protein p115, which regulates ER-Golgi trafficking steps, a ~60 residue coiled-coil domain with homology to t-SNAREs interacts with several SNARE proteins and promotes trans SNARE complex formation (Shorter et al., 2002). Tomosyn, an inhibitory protein for vesicle exocytosis, harbors a VAMP motif that may interfere with SNARE complex formation (Yamamoto et al., 2009). Other tethering factor complexes involved in membrane fusion events (exocyst, COG, GARP, Dsl1p) contain subunits that bind SNAREs via helical domains (Sztul and Lupashin, 2009). Regions in these tethering factor subunits exhibit an evolutionary relationship to regions in the CAPS and Munc13 proteins. The helix in the CAPS MHD1 identified here as part of the SNARE-binding domain is at the N-terminal end of the tethering factor homology region (Pei, 2009).

While sequences in MHD1 were necessary for CAPS binding to SNAREs and for CAPS activity in vesicle exocytosis, these sequences were not sufficient for CAPS activity. This indicates that additional regions of CAPS are needed to integrate its SNARE-binding activity with its function in vesicle exocytosis. Indeed, the PI(4,5)P2-binding PH domain of CAPS was essential for CAPS stimulation of SNARE complex formation and SNARE-dependent liposome fusion (James et al., 2009). CAPS appears to utilize both membrane interactions and SNARE binding to execute its function. Other domains in CAPS also play essential but poorly-understood roles (Grishanin et al., 2002; Nojiri et al., 2009; Speese et al., 2007).

The current work expands our understanding of the function of domains in CAPS and provides a plausible explanation for how CAPS could promote SNARE complex formation in vesicle priming. Following plasma membrane recruitment via PH domain interactions with acidic phospholipids, t-SNARE-binding sequences in the DAMH domain could engage syntaxin-1 and SNAP-25 to organize t-SNARE complexes. Subsequently, binding to t-SNAREs by the DAMH domain may be displaced by the interaction of overlapping v-SNARE-binding sequences in the DAMH domain with VAMP-2. This interaction could facilitate the entry of VAMP-2 into t-SNARE complexes possibly accompanied by CAPS dissociation. This speculative model for the mechanism of CAPS function will require further assessment.

EXPERIMENTAL PROCEDURES

Preparation of liposomes

Proteoliposomes were formed by co-micellization or by the extrusion method (James et al., 2008; Mayer et al., 1986; Weber et al., 1998). For co-micellization, a lipid film with 1.5 μmoles DOPC and 0.25 μmoles DOPS was resuspended with 500 μl SNARE proteins in elution buffer (25 mM HEPES-KOH pH 7.4, 100 mM KCl, 50mM imidazole-OAc, pH 7.4, 1.0% β-octylglucoside). Lipid mixtures contained 2 μCi of [3H]-1,2-dipalmitoyl phosphatidylcholine (NEN, Cambridge, UK) to determine lipid recoveries and to standardize binding reactions. Proteoliposomes were dialyzed overnight at 4°C in reconstitution buffer (25mM HEPES-KOH pH 7.4, 100mM KCl, 10% (w/v) glycerol, 1mM DTT) stirring with Bio-beads® (BIO-RAD), mixed with an equal volume of 80% Nycodenz® (a.k.a. Accudenz®) (Accurate Chemical and Scientific Corp, NY, USA), overlaid with 30% and 0% Accudenz in reconstitution buffer without glycerol, and centrifuged at 45,000 rpm for 4 h at 4°C in a SW 50.1 rotor. Proteoliposomes at the 0-30% Accudenz interface were collected and flash frozen. For the extrusion method, a lipid film containing 4.5 μmoles DOPC and 0.75 μmoles DOPS was resuspended in 300 μl of reconstitution buffer, vortexed, frozen-thawed 5 times, and extruded (MiniExtruder, Avanti Polar Lipids) with 32 passes through a 0.1 μm Nuclepore® track-etched polycarbonate membrane filter (Whatman) to produce 100 nm diameter proteoliposomes. Aliquots were mixed quickly with 2 volumes of SNARE proteins in elution buffer, gently shaken for 30 min, diluted with 1 volume reconstitution buffer, dialyzed and separated by gradient centrifugation as above. SNARE protein content was assessed by SDS-PAGE and Coomassie staining. Proteoliposomes by extrusion had ~5096 copies/μm2 (~160 copies/liposome) of syntaxin/SNAP-25 or ~11,465 copies/μm2 (~360 copies/liposome) of VAMP-2. Extruded liposomes with thrombin-cleavable syntaxin-1A/SNAP-25B were treated with human thrombin (Sigma-Aldrich, St. Louis, MO) at 0.02 units/ml for 2h at room temperature and terminated with 2 mM 4-(2-aminoethyl) benzenesulfonylfluoride (Calbiochem Corp) as described (Parlati et al., 1999). SNAP-25 was crosslinked to extruded liposomes composed of DOPC:DOPS:MPB-PE (80:15:5) (Avanti Polar Lipids) by incubation for 1h in buffers with TCEP instead of DTT and quenched with 1mM β-mercaptoethanol.

Liposome binding assay

Protein-free liposomes or liposomes containing syntaxin-1A, SNAP-25, syntaxin-1A/SNAP-25, or VAMP-2 were in reconstitution buffer without glycerol were incubated with CAPS or CAPS fusion proteins at room temperature for 30 min. Bound and free proteins were separated by Accudenz gradient centrifugation at 45,000 rpm for 1-4h at 4 ºC but maximal recovery was achieved within 30 min. Top fractions were collected and 20,000 DPM recovered liposomes were run on SDS-PAGE for staining with SYPRO® Ruby (Invitrogen) or for Western blotting with a polyclonal CAPS antibody, a monoclonal GST antibody (Sigma-Aldrich, St. Louis, MO), a polyclonal MBP antibody (New England Biolabs, Ipswich, MA), a monoclonal syntaxin-1A antibody (Sigma-Aldrich, St. Louis, MO), a polyclonal VAMP-2 antibody, or a polyclonal SNAP-25 antibody (Life Span Biosciences, Seattle, WA). Autoradiograms were quantified with a Molecular Dynamics Densitometer using ImageQuant software. SYPRO® Ruby-stained gels were analyzed on a Typhoon 9410 (Amersham Biosciences, Piscataway, NJ).

Secretion assays

A permeable PC12 cell assay was used to assess CAPS activity with norepinephrine release measured by rotating disc electrode voltammetry (Grishanin et al., 2004). For intact cell assays, PC12 cells were transfected to express brain-derived neurotrophic factor (BDNF)-enhanced green fluorescent protein (EGFP) as described (Lynch et al., 2008). Images were acquired on a Nikon TE2000 microscope fitted for TIRF with an Apo TIRF 100X NA 1.45 objective lens with a CoolSNAP-ES Digital Monochrome CCD Camera or an Evolve Digital Monochrome emCCD Camera (Photometrics) controlled by Metamorph software (Universal Imaging Corp). EGFP was excited at 488 nm and tagRFP or mKate2 at 543 nm. Two channel images were acquired with a dual view imaging system (Optical Insights). CAPS was down-regulated by transfection with an shRNA plasmid (Nojiri et al., 2009) targeting nucleotides 3839-3866 of CAPS mRNA. CAPS rescue constructs containing four silent mutations in the target sequence were subcloned into tagRFP-containing plasmids (Evrogen, Moscow, Russia). Colocalizations were calculated using ImageJ software (Wayne Rasband, NIH) with a colocalization threshold plugin (Tony Collins) and are reported as the thresholded Mander’s split colocalization coefficient (1.0 is perfect colocalization; 0.0 is no colocalization.

Other methods

Description of materials, DNA constructs, recombinant protein expression and purification can be found in Supplemental Information.

Supplementary Material

01

Acknowledgments

This work was supported by a grant from the NIH (DK040428) to T.F.J.M., fellowship support from the American Heart Association to D.J.J., and NIH training grant (GM007507) support to C.K. The authors acknowledge former students in the Martin laboratory, M. Nojiri and S. Doughman, for contributions to early stages of the work.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Ann K, Kowalchyk JA, Loyet KM, Martin TF. Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis. The Journal of biological chemistry. 1997;272:19637–19640. doi: 10.1074/jbc.272.32.19637. [DOI] [PubMed] [Google Scholar]
  2. Basu J, Shen N, Dulubova I, Lu J, Guan R, Guryev O, Grishin NV, Rosenmund C, Rizo J. A minimal domain responsible for Munc13 activity. Nature structural & molecular biology. 2005;12:1017–1018. doi: 10.1038/nsmb1001. [DOI] [PubMed] [Google Scholar]
  3. Betz A, Okamoto M, Benseler F, Brose N. Direct interaction of the rat unc-13 homologue Munc13-1 with the N terminus of syntaxin. The Journal of biological chemistry. 1997;272:2520–2526. doi: 10.1074/jbc.272.4.2520. [DOI] [PubMed] [Google Scholar]
  4. Daily NJ, Boswell KL, James DJ, Martin TF. Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion. The Journal of biological chemistry. 2010;285:35320–35329. doi: 10.1074/jbc.M110.145169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elhamdani A, Martin TF, Kowalchyk JA, Artalejo CR. Ca(2+)-dependent activator protein for secretion is critical for the fusion of dense-core vesicles with the membrane in calf adrenal chromaffin cells. J Neurosci. 1999;19:7375–7383. doi: 10.1523/JNEUROSCI.19-17-07375.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellena JF, Liang B, Wiktor M, Stein A, Cafiso DS, Jahn R, Tamm LK. Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:20306–20311. doi: 10.1073/pnas.0908317106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fujita Y, Xu A, Xie L, Arunachalam L, Chou TC, Jiang T, Chiew SK, Kourtesis J, Wang L, Gaisano HY, et al. Ca2+-dependent activator protein for secretion 1 is critical for constitutive and regulated exocytosis but not for loading of transmitters into dense core vesicles. The Journal of biological chemistry. 2007;282:21392–21403. doi: 10.1074/jbc.M703699200. [DOI] [PubMed] [Google Scholar]
  8. Grishanin RN, Klenchin VA, Loyet KM, Kowalchyk JA, Ann K, Martin TF. Membrane association domains in Ca2+-dependent activator protein for secretion mediate plasma membrane and dense-core vesicle binding required for Ca2+-dependent exocytosis. The Journal of biological chemistry. 2002;277:22025–22034. doi: 10.1074/jbc.M201614200. [DOI] [PubMed] [Google Scholar]
  9. Grishanin RN, Kowalchyk JA, Klenchin VA, Ann K, Earles CA, Chapman ER, Gerona RR, Martin TF. CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein. Neuron. 2004;43:551–562. doi: 10.1016/j.neuron.2004.07.028. [DOI] [PubMed] [Google Scholar]
  10. Hammarlund M, Watanabe S, Schuske K, Jorgensen EM. CAPS and syntaxin dock dense core vesicles to the plasma membrane in neurons. The Journal of cell biology. 2008;180:483–491. doi: 10.1083/jcb.200708018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jahn R, Scheller RH. SNAREs--engines for membrane fusion. Nature reviews. 2006;7:631–643. doi: 10.1038/nrm2002. [DOI] [PubMed] [Google Scholar]
  12. James DJ, Khodthong C, Kowalchyk JA, Martin TF. Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion. The Journal of cell biology. 2008;182:355–366. doi: 10.1083/jcb.200801056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. James DJ, Kowalchyk J, Daily N, Petrie M, Martin TF. CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:17308–17313. doi: 10.1073/pnas.0900755106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koch H, Hofmann K, Brose N. Definition of Munc13-homology-domains and characterization of a novel ubiquitously expressed Munc13 isoform. The Biochemical journal. 2000;349:247–253. doi: 10.1042/0264-6021:3490247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Liu Y, Schirra C, Stevens DR, Matti U, Speidel D, Hof D, Bruns D, Brose N, Rettig J. CAPS facilitates filling of the rapidly releasable pool of large dense-core vesicles. J Neurosci. 2008;28:5594–5601. doi: 10.1523/JNEUROSCI.5672-07.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Loyet KM, Kowalchyk JA, Chaudhary A, Chen J, Prestwich GD, Martin TF. Specific binding of phosphatidylinositol 4,5-bisphosphate to calcium-dependent activator protein for secretion (CAPS), a potential phosphoinositide effector protein for regulated exocytosis. The Journal of biological chemistry. 1998;273:8337–8343. doi: 10.1074/jbc.273.14.8337. [DOI] [PubMed] [Google Scholar]
  17. Lynch KL, Gerona RR, Kielar DM, Martens S, McMahon HT, Martin TF. Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion. Molecular biology of the cell. 2008;19:5093–5103. doi: 10.1091/mbc.E08-03-0235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ma C, Li W, Xu Y, Rizo J. Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex. Nature structural & molecular biology. 2011 doi: 10.1038/nsmb.2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Madison JM, Nurrish S, Kaplan JM. UNC-13 interaction with syntaxin is required for synaptic transmission. Curr Biol. 2005;15:2236–2242. doi: 10.1016/j.cub.2005.10.049. [DOI] [PubMed] [Google Scholar]
  20. Martin TF, Kowalchyk JA. Docked secretory vesicles undergo Ca2+-activated exocytosis in a cell-free system. The Journal of biological chemistry. 1997;272:14447–14453. doi: 10.1074/jbc.272.22.14447. [DOI] [PubMed] [Google Scholar]
  21. Mayer LD, Hope MJ, Cullis PR. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochimica et biophysica acta. 1986;858:161–168. doi: 10.1016/0005-2736(86)90302-0. [DOI] [PubMed] [Google Scholar]
  22. Nojiri M, Loyet KM, Klenchin VA, Kabachinski G, Martin TF. CAPS activity in priming vesicle exocytosis requires CK2 phosphorylation. The Journal of biological chemistry. 2009;284:18707–18714. doi: 10.1074/jbc.M109.017483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olsen HL, Hoy M, Zhang W, Bertorello AM, Bokvist K, Capito K, Efanov AM, Meister B, Thams P, Yang SN, et al. Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:5187–5192. doi: 10.1073/pnas.0931282100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Parlati F, Weber T, McNew JA, Westermann B, Sollner TH, Rothman JE. Rapid and efficient fusion of phospholipid vesicles by the alpha-helical core of a SNARE complex in the absence of an N-terminal regulatory domain. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:12565–12570. doi: 10.1073/pnas.96.22.12565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pei J, Ma C, Rizo J, Grishin NV. Remote homology between Munc13 MUN domain and vesicle tethering complexes. J Mol Biol. 2009;391:509–517. doi: 10.1016/j.jmb.2009.06.054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rettig J, Neher E. Emerging roles of presynaptic proteins in Ca++-triggered exocytosis. Science (New York, NY. 2002;298:781–785. doi: 10.1126/science.1075375. [DOI] [PubMed] [Google Scholar]
  27. Sato M, Mori Y, Matsui T, Aoki R, Oya M, Yanagihara Y, Fukuda M, Tsuboi T. Role of the polybasic sequence in the Doc2alpha C2B domain in dense-core vesicle exocytosis in PC12 cells. Journal of neurochemistry. 2010;114:171–181. doi: 10.1111/j.1471-4159.2010.06739.x. [DOI] [PubMed] [Google Scholar]
  28. Shorter J, Beard MB, Seemann J, Dirac-Svejstrup AB, Warren G. Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115. The Journal of cell biology. 2002;157:45–62. doi: 10.1083/jcb.200112127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Speese S, Petrie M, Schuske K, Ailion M, Ann K, Iwasaki K, Jorgensen EM, Martin TF. UNC-31 (CAPS) is required for dense-core vesicle but not synaptic vesicle exocytosis in Caenorhabditis elegans. J Neurosci. 2007;27:6150–6162. doi: 10.1523/JNEUROSCI.1466-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Speidel D, Salehi A, Obermueller S, Lundquist I, Brose N, Renstrom E, Rorsman P. CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic beta cells. Cell metabolism. 2008;7:57–67. doi: 10.1016/j.cmet.2007.11.009. [DOI] [PubMed] [Google Scholar]
  31. Stevens DR, Rettig J. The Ca(2+)-dependent activator protein for secretion CAPS: do I dock or do I prime? Molecular neurobiology. 2009;39:62–72. doi: 10.1007/s12035-009-8052-5. [DOI] [PubMed] [Google Scholar]
  32. Stevens DR, Wu ZX, Matti U, Junge HJ, Schirra C, Becherer U, Wojcik SM, Brose N, Rettig J. Identification of the minimal protein domain required for priming activity of Munc13-1. Curr Biol. 2005;15:2243–2248. doi: 10.1016/j.cub.2005.10.055. [DOI] [PubMed] [Google Scholar]
  33. Sutton RB, Fasshauer D, Jahn R, Brunger AT. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature. 1998;395:347–353. doi: 10.1038/26412. [DOI] [PubMed] [Google Scholar]
  34. Sztul E, Lupashin V. Role of vesicle tethering factors in the ER-Golgi membrane traffic. FEBS letters. 2009;583:3770–3783. doi: 10.1016/j.febslet.2009.10.083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Takahashi N, Hatakeyama H, Okado H, Noguchi J, Ohno M, Kasai H. SNARE conformational changes that prepare vesicles for exocytosis. Cell metabolism. 2010;12:19–29. doi: 10.1016/j.cmet.2010.05.013. [DOI] [PubMed] [Google Scholar]
  36. Walent JH, Porter BW, Martin TF. A novel 145 kd brain cytosolic protein reconstitutes Ca(2+)-regulated secretion in permeable neuroendocrine cells. Cell. 1992;70:765–775. doi: 10.1016/0092-8674(92)90310-9. [DOI] [PubMed] [Google Scholar]
  37. Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Sollner TH, Rothman JE. SNAREpins: minimal machinery for membrane fusion. Cell. 1998;92:759–772. doi: 10.1016/s0092-8674(00)81404-x. [DOI] [PubMed] [Google Scholar]
  38. Wiederhold K, Kloepper TH, Walter AM, Stein A, Kienle N, Sorensen JB, Fasshauer D. A coiled-coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex. The Journal of biological chemistry. 2010 Apr 20; doi: 10.1074/jbc.M110.105148. [Epub ahead of print] [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wojcik SM, Brose N. Regulation of membrane fusion in synaptic excitation-secretion coupling: speed and accuracy matter. Neuron. 2007;55:11–24. doi: 10.1016/j.neuron.2007.06.013. [DOI] [PubMed] [Google Scholar]
  40. Yamamoto Y, Mochida S, Kurooka T, Sakisaka T. Reciprocal intramolecular interactions of tomosyn control its inhibitory activity on SNARE complex formation. The Journal of biological chemistry. 2009;284:12480–12490. doi: 10.1074/jbc.M807182200. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS311491-supplement-01.doc (518.5KB, doc)

RESOURCES