Habc Domain Research Articles

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts.

SNARE proteins drive membrane fusion as their core domains zipper into a parallel four-helix bundle1,2. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/ α-SNAP3,4 to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains5. Previous structures of the NSF-α-SNAP-SNARE complex revealed SNARE domain threaded through the D1 ATPase ring6, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems7. Here, in vivo mass-spectrometry reveals N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. Cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition shows SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.

SM protein Sly1 and a SNARE Habc domain promote membrane fusion through multiple mechanisms.

SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore.

Syntaxin clusters and cholesterol affect the mobility of Syntaxin1a

Syntaxin1a (Syx1a) is essential for stimulated exocytosis in neuroendocrine cells. The vesicle docking process involves the formation of nanoscale Syx1a domains on the plasma membrane and the Syx1a clusters disintegrate during the fusion process. Syx1a nanodomains are static yet Syx1a molecules dynamically enter and leave the domains; the process by which these clusters maintain this balance is unclear. In this work, the dynamics of the Syx1a molecules is elucidated relative to the cluster position through a labeling strategy that allows both the bulk position of the Syx clusters to be visualized concurrent with the trajectories of single Syx1a molecules on the surface of PC12 cells. Single Syx1a molecules were tracked in time relative to cluster positions to decipher how Syx1a moves within a cluster and when clusters are not present. Syx1a is mobile on the plasma membrane, more mobile at the center of clusters, and less mobile near the edges of clusters; this depends on the presence of the N-terminal Habc domain and cholesterol, which are essential for proper exocytosis. Simulations of the dynamics observed at clusters support a model where clusters are maintained by a large cage (r = 100 nm) within which Syx1a remains highly mobile within the cluster (r = 50 nm). The depletion of cholesterol dramatically reduces the mobility of Syx1a within clusters and less so over the rest of the plasma membrane. This suggests that fluidity of Syx1a supramolecular clusters is needed for function.

The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain.

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering, and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 s half-life) colocalizing partially with the Golgi markers RAB1, Sed5, and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycle back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments, Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing a stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases the binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that, unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.

Syntaxin17 contributes to obesity cardiomyopathy through promoting mitochondrial Ca2+ overload in a Parkin-MCUb-dependent manner

ObjectiveUncorrected obesity is accompanied by unfavorable structural and functional changes in the heart, known as obesity cardiomyopathy. Recent evidence has revealed a crucial role for mitochondria-associated endoplasmic reticulum membranes (MAMs) in obesity-induced cardiac complication. Syntaxin 17 (STX17) serves as a scaffolding molecule localized on MAMs although its role in obesity heart complication remains elusive. Methods and materialsThis study examined the role of STX17 in MAMs and mitochondrial Ca2+ homeostasis in HFD-induced obesity cardiomyopathy using tamoxifen-induced cardiac-specific STX17 knockout (STX17cko) and STX17 overexpression mice using intravenously delivered recombinant adeno-associated virus serotype-9 (AAV9-cTNT-STX17). ResultsSTX17 levels were significantly elevated in plasma from obese patients and heart tissues of HFD-fed mice. Our data revealed that cardiac STX17 knockout alleviated cardiac remodeling and dysfunction in obese hearts without eliciting any notable effect itself, while STX17 overexpression aggravated cardiac dysfunction in obese mice. STX17 deletion and STX17 overexpression annihilated and aggravated, respectively, HFD-induced oxidative stress (O2− production) and mitochondrial injury in the heart. Furthermore, STX17 transfection facilitated obesity-induced MAMs formation in cardiomyocytes and evoked excess mitochondrial Ca2+ influx, dependent upon interaction with mitochondrial Ca2+ uniporter dominant negative β (MCUb) through Habc domain. Our data also suggested that STX17 promoted ubiquitination and degradation of MCUb through the E3 ligase parkin in the face of palmitate challenging. ConclusionTaken together, our results identified a novel role for STX17 in facilitating obesity-induced MAMs formation, and subsequently mitochondrial Ca2+ overload, mitochondrial O2− accumulation, lipid peroxidation, resulting in cardiac impairment. Our findings denoted therapeutic promises of targeting STX17 in obesity.

Interspecies complementation identifies a pathway to assemble SNAREs

SummaryUnc18 and SNARE proteins form the core of the membrane fusion complex at synapses. To understand the functional interactions within the core machinery, we adopted an “interspecies complementation” approach in Caenorhabditis elegans. Substitutions of individual SNAREs and Unc18 proteins with those from yeast fail to rescue fusion. However, synaptic transmission could be restored in worm-yeast chimeras when two key interfaces were present: an Habc-Unc18 contact site and an Unc18-SNARE motif contact site. A constitutively open form of Unc18 bypasses the requirement for the Habc-Unc18 interface. These data suggest that the Habc domain of syntaxin is required for Unc18 to adopt an open conformation; open Unc18 then templates SNARE complex formation. Finally, we demonstrate that the SNARE and Unc18 machinery in the nematode C. elegans can be replaced by yeast proteins and still carry out synaptic transmission, pointing to the deep evolutionary conservation of these two interfaces.

SNARE SYP132 mediates divergent traffic of plasma membrane H+-ATPase AHA1 and antimicrobial PR1 during bacterial pathogenesis.

The vesicle trafficking SYNTAXIN OF PLANTS132 (SYP132) drives hormone-regulated endocytic traffic to suppress the density and function of plasma membrane (PM) H+-ATPases. In response to bacterial pathogens, it also promotes secretory traffic of antimicrobial pathogenesis-related (PR) proteins. These seemingly opposite actions of SYP132 raise questions about the mechanistic connections between the two, likely independent, membrane trafficking pathways intersecting plant growth and immunity. To study SYP132 and associated trafficking of PM H+-ATPase 1 (AHA1) and PATHOGENESIS-RELATED PROTEIN1 (PR1) during pathogenesis, we used the virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacteria for infection of Arabidopsis (Arabidopsis thaliana) plants. SYP132 overexpression suppressed bacterial infection in plants through the stomatal route. However, bacterial infection was enhanced when bacteria were infiltrated into leaf tissue to bypass stomatal defenses. Tracking time-dependent changes in native AHA1 and SYP132 abundance, cellular distribution, and function, we discovered that bacterial pathogen infection triggers AHA1 and SYP132 internalization from the plasma membrane. AHA1 bound to SYP132 through its regulatory SNARE Habc domain, and these interactions affected PM H+-ATPase traffic. Remarkably, using the Arabidopsis aha1 mutant, we discovered that AHA1 is essential for moderating SYP132 abundance and associated secretion of PR1 at the plasma membrane for pathogen defense. Thus, we show that during pathogenesis SYP132 coordinates AHA1 with opposing effects on the traffic of AHA1 and PR1.

The Habc domain of syntaxin 3 is a ubiquitin binding domain

Syntaxins are a family of membrane-anchored SNARE proteins that are essential components required for membrane fusion in eukaryotic intracellular membrane trafficking pathways. Syntaxins contain an N-terminal regulatory domain, termed the Habc domain that is not highly conserved at the primary sequence level but folds into a three-helix bundle that is structurally conserved among family members. The syntaxin Habc domain has previously been found to be structurally very similar to the GAT domain present in GGA family members and related proteins that are otherwise completely unrelated to syntaxins. Because the GAT domain has been found to be a ubiquitin binding domain we hypothesized that the Habc domain of syntaxins may also bind to ubiquitin. Here, we report that the Habc domain of syntaxin 3 (Stx3) indeed binds to monomeric ubiquitin with low affinity. This domain binds efficiently to K63-linked poly-ubiquitin chains within a narrow range of chain lengths but not to K48-linked poly-ubiquitin chains. Other syntaxin family members also bind to K63-linked poly-ubiquitin chains but with different chain length specificities. Molecular modeling suggests that residues of the GGA3-GAT domain known to be important for ionic and hydrophobic interactions with ubiquitin may have equivalent, conserved residues within the Habc domain of Stx3. We conclude that the syntaxin Habc domain and the GAT domain are both structurally and functionally related, and likely share a common ancestry despite sequence divergence. Binding of Ubiquitin to the Habc domain may regulate the function of syntaxins in membrane fusion or may suggest additional functions of this protein family.

The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation.

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM-Qa-SNARE complex, Vps45-Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45-Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

Epilepsy-causing STX1B mutations translate altered protein functions into distinct phenotypes in mouse neurons.

Syntaxin 1B (STX1B) is a core component of the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that is critical for the exocytosis of synaptic vesicles in the presynapse. SNARE-mediated vesicle fusion is assisted by Munc18-1, which recruits STX1B in the auto-inhibited conformation, while Munc13 catalyses the fast and efficient pairing of helices during SNARE complex formation. Mutations within the STX1B gene are associated with epilepsy. Here we analysed three STX1B mutations by biochemical and electrophysiological means. These three paradigmatic mutations cause epilepsy syndromes of different severity, from benign fever-associated seizures in childhood to severe epileptic encephalopathies. An insertion/deletion (K45/RMCIE, L46M) mutation (STX1BInDel), causing mild epilepsy and located in the early helical Habc domain, leads to an unfolded protein unable to sustain neurotransmission. STX1BG226R, causing epileptic encephalopathies, strongly compromises the interaction with Munc18-1 and reduces expression of both proteins, the size of the readily releasable pool of vesicles, and Ca2+-triggered neurotransmitter release when expressed in STX1-null neurons. The mutation STX1BV216E, also causing epileptic encephalopathies, only slightly diminishes Munc18-1 and Munc13 interactions, but leads to enhanced fusogenicity and increased vesicular release probability, also in STX1-null neurons. Even though the synaptic output remained unchanged in excitatory hippocampal STX1B+/- neurons exogenously expressing STX1B mutants, the manifestation of clear and distinct molecular disease mechanisms by these mutants suggest that certain forms of epilepsies can be conceptualized by assigning mutations to structurally sensitive regions of the STX1B-Munc18-1 interface, translating into distinct neurophysiological phenotypes.

Multiple features within the syntaxin Sed5p mediate its Golgi localization

Protein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi-the Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells, the syntaxin SNARE Sed5p oversees membrane fusion events. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the steady-state localization of Sed5p to the Golgi appears to be primarily conformation-based relying on intra-molecular associations between the Habc domain and SNARE-motif while its tribasic COPI-coatomer binding motif plays a role in intra-Golgi retention.

The molecular requirements for the G protein betagamma‐SNARE interaction

Inhibitory G‐protein coupled receptors (GPCRs) can attenuate the secretion of hormones and neurotransmitters in an Ca2+‐dependent fashion. It has been shown that one mechanism for this inhibition involves the direct interaction of G‐protein βγ dimers (Gβγ) with the soluble N‐ethylmaleimide sensitive attachment protein receptor (SNARE) proteins syntaxin1A (Stx1A) and SNAP‐25. We have previously shown that Gβγ competes with the calcium sensor synaptotagmin 1 (Syt1) for binding sites upon SNARE proteins in a Ca2+ dependent manner. The goal of this study is to determine the individual residues required for the Gβγ‐SNARE interaction. To investigate the molecular basis for this interaction, prior studies performed by our group utilizing a “peptide mapping” approach have shown that both the N‐terminal and the C‐terminal domains of SNAP‐25 are essential for the interaction of Gβγ with SNAP‐25. Here, utilizing a similar approach, we demonstrate that the N‐terminal Habc domain is essential for the interaction of Gβγ with Stx1A. The interaction is mediated through two positively‐charged residues: K55 and R125. Mutation of these residues to Ala abolishes Gβγ binding in vitro, while retaining the ability to bind Syt1. We anticipate that these studies will enhance our understanding of the GPCR‐mediated regulatory mechanisms of exocytosis.

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion.

An N-Terminal Missense Mutation in STX11 Causative of FHL4 Abrogates Syntaxin-11 Binding to Munc18-2

Familial hemophagocytic lymphohistiocytosis (FHL) is an often-fatal hyperinflammatory disorder caused by autosomal recessive mutations in PRF1, UNC13D, STX11, and STXBP2. We identified a homozygous STX11 mutation, c.173T > C (p.L58P), in three patients presenting clinically with hemophagocytic lymphohistiocytosis from unrelated Pakistani families. The mutation yields an amino acid substitution in the N-terminal Habc domain of syntaxin-11 and resulted in defective natural killer cell degranulation. Notably, syntaxin-11 expression was decreased in patient cells. However, in an ectopic expression system, syntaxin-11 L58P was expressed at levels comparable to wild-type syntaxin-11, but did not bind Munc18-2. Moreover, another N-terminal syntaxin-11 mutant, R4A, also did not bind Munc18-2. Thus, we have identified a novel missense STX11 mutation causative of FHL type 4. The syntaxin-11 R4A and L58P mutations reveal that both the N-terminus and Habc domain of syntaxin-11 are required for binding to Munc18-2, implying similarity to the dynamic binary binding of neuronal syntaxin-1 to Munc18-1.

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein Complex with the t-SNARE Syntaxin 6

The Golgi-Associated Retrograde Protein (GARP) complex is a tethering factor involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein, we report the structural basis for the interaction of the human Ang2 subunit of GARP with the Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of the Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a binding mode in which a dityrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction.

Allosteric Control of Syntaxin 1a by Munc18-1: Characterization of the Open and Closed Conformations of Syntaxin

Syntaxin 1a is a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor protein (SNARE) that contains an H3 domain (SNARE motif) and a regulatory Habc domain. These regions associate to produce a closed state, which is generally thought to suppress assembly of syntaxin into the SNARE complex. However, the molecular nature of the closed and open states of syntaxin is not well defined. Here, we use electron paramagnetic resonance spectroscopy to characterize conformational exchange in syntaxin. The data indicate that the H3 segment is in equilibrium between ordered and disordered states that have significant populations. In solution, the central region of the H3 segment is positioned close to the Habc domain and the configuration of syntaxin 1a is dominated by a closed state. However, an open state is enhanced in full-length membrane reconstituted syntaxin. Munc18-1 binding alters the equilibrium along H3 to favor the ordered, folded state. Munc18 also suppresses the minor open population and narrows the distance distributions between H3 and Habc. The allosteric control exhibited by Munc18 on the H3 segment and the suppression of the minor open component may both play a role in regulating membrane fusion by controlling the assembly of syntaxin into the SNARE complex.

Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H(abc) domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.

Sec1/Munc18 Protein Stabilizes Fusion-Competent Syntaxin for Membrane Fusion in Arabidopsis Cytokinesis

Intracellular membrane fusion requires complexes of syntaxins with other SNARE proteins and regulatory Sec1/Munc18 (SM) proteins. In membrane fusion mediating, e.g., neurotransmitter release or glucose-stimulated insulin secretion in mammals, SM proteins preferentially interact with the inactive closed, rather than the active open, conformation of syntaxin or with the assembled SNARE complex. Other membrane fusion processes such as vacuolar fusion in yeast involve like membranes carrying cis-SNARE complexes, and the role of SM protein is unknown. We investigated syntaxin-SM protein interaction in membrane fusion of Arabidopsis cytokinesis, which involves cytokinesis-specific syntaxin KNOLLE and SM protein KEULE. KEULE interacted with an open conformation of KNOLLE that complemented both knolle and keule mutants. This interaction occurred at the cell division plane and required the KNOLLE linker sequence between helix Hc and SNARE domain. Our results suggest that in cytokinesis, SM protein stabilizes the fusion-competent open form of syntaxin, thereby promoting trans-SNARE complex formation.

The Allosteric Modulation of Syntaxin 1A by Munc18: Characterization of the Open and Closed Conformations of Syntaxin

Syntaxin 1A is a plasma membrane SNARE that contains a SNARE motif (H3 domain) and a regulatory Habc domain. It is generally believed that syntaxin can exist in both an open and closed conformation, where the H3 domain is either free or associated with the Habc domain. Munc18, a regulatory SM protein was discovered on the basis of its high affinity to syntaxin. It is believed to arrest membrane fusion by locking syntaxin 1A into a closed state so that it cannot assemble into a SNARE complex with SNAP25 and synaptobrevin. In the present work, we use continuous wave and pulse EPR to characterize the open and closed states of syntaxin 1A. The data indicate that the H3 motif is always closely associated with the Habc domain, irrespective of the presence of munc18. Segments of the H3 motif are in conformational exchange between ordered and disordered states and munc18 acts to shift this conformational equilibrium to the ordered state. The results indicate that munc18 functions as an allosteric regulator of syntaxin 1A, and we postulate that this regulation functions to control the dynamics of those segments of syntaxin that participate in SNARE recognition and assembly.

Munc13 Homology Domain-1 in CAPS/UNC31 Mediates SNARE Binding Required for Priming Vesicle Exocytosis

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-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 identify 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 Ca(2+)-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.