SNAREpins are functionally resistant to disruption by NSF and alphaSNAP

SNARE (SNAP [soluble NSF {N-ethylmaleimide–sensitive fusion protein} attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn''t NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.     

Novel putative targets of N-ethylmaleimide sensitive fusion protein (NSF) and alpha/beta soluble NSF attachment proteins (SNAPs) include the Pak-binding nucleotide exchange factor betaPIX

N-ethylmaleimide sensitive fusion protein (NSF) is a chaperone that plays a crucial role in the fusion of vesicles with target membranes. NSF mediates the ATP-consuming dissociation of a core protein complex that assembles during vesicle fusion and it thereby recharges the fusion machinery to perform multiple rounds of fusion. The binding of NSF to the core complex is mediated by co-chaperones named soluble NSF attachment proteins (SNAPs), for which three isoforms (alpha, beta and gamma) are known. Here, we sought to identify novel targets of the NSF-SNAP complex. A yeast two-hybrid screen using the brain specific betaSNAP isoform as bait revealed, as expected, NSF and several isoforms of the SNARE protein syntaxin as interactors. In addition, three isoforms of the reticulon protein family and two isoforms of BNIP3 interacted with betaSNAP. A yeast two-hybrid screen using NSF as bait identified Rab11-FIP3 and the Pak-binding nucleotide exchange factor betaPIX as putative binding partners. betaPIX interacts with recombinant NSF in co-sedimentation assays and the two proteins may be co-immunoprecipitated. A leucine zipper (LZ) motif within the C-terminus of betaPIX mediates binding to NSF; however, this fragment of betaPIX does not exhibit dominant negative effects in a cellular assay. In summary, our results support the evolving view that NSF has numerous targets in addition to conventional SNARE complexes.     

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca²⁺ levels, and a major Ca²⁺ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca²⁺ entry to vesicle fusion via Ca²⁺-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.     

Cryo-EM structure of SNAP-SNARE assembly in 20S particle

N-ethylmaleimide-sensitive factor (NSF) and α soluble NSF attachment proteins (α-SNAPs) work together within a 20S particle to disassemble and recycle the SNAP receptor (SNARE) complex after intracellular membrane fusion. To understand the disassembly mechanism of the SNARE complex by NSF and α-SNAP, we performed single-particle cryo-electron microscopy analysis of 20S particles and determined the structure of the α-SNAP-SNARE assembly portion at a resolution of 7.35 Å. The structure illustrates that four α-SNAPs wrap around the single left-handed SNARE helical bundle as a right-handed cylindrical assembly within a 20S particle. A conserved hydrophobic patch connecting helices 9 and 10 of each α-SNAP forms a chock protruding into the groove of the SNARE four-helix bundle. Biochemical studies proved that this structural element was critical for SNARE complex disassembly. Our study suggests how four α-SNAPs may coordinate with the NSF to tear the SNARE complex into individual proteins.     

SNARE-mediated membrane traffic modulates RhoA-regulated focal adhesion formation

In the present study, we examined the role of soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic in the formation of focal adhesions during cell spreading. CHO-K1 cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (E329Q-NSF) were unable to spread as well as control cells and they formed focal adhesions (FAs) that were larger than those in control cells. FA formation was impaired in cells transfected with a dominant-negative form of RhoA, but, significantly, not in cells simultaneously expressing dominant-negative NSF. Treatment of E329Q-NSF-expressing cells with the ROCK inhibitor Y-27632 did inhibit FA formation. The results are consistent with a model of cell adhesion in which SNARE-mediated membrane traffic is required for both the elaboration of lamellipodia and the modulation of biochemical signals that control RhoA-mediated FA assembly.     

Probing the structural dynamics of the SNARE recycling machine based on coarse‐grained modeling

Membrane fusion in eukaryotes is driven by the formation of a four‐helix bundle by three SNARE proteins. To recycle the SNARE proteins, they must be disassembled by the ATPase NSF and four SNAP proteins which together form a 20S supercomplex. Recently, the first high‐resolution structures of the NSF (in both ATP and ADP state) and 20S (in four distinct states termed I, II, IIIa, and IIIb) were solved by cryo‐electron microscopy (cryo‐EM), which have paved the way for structure‐driven studies of the SNARE recycling mechanism. To probe the structural dynamics of SNARE disassembly at amino‐acid level of details, a systematic coarse‐grained modeling based on an elastic network model and related analyses were performed. Our normal mode analysis of NSF, SNARE, and 20S predicted key modes of collective motions that partially account for the observed structural changes, and illuminated how the SNARE complex can be effectively destabilized by untwisting and bending motions of the SNARE complex driven by the amino‐terminal domains of NSF in state II. Our flexibility analysis identified regions with high/low flexibility that coincide with key functional sites (such as the NSF‐SNAPs‐SNARE binding sites). A subset of hotspot residues that control the above collective motions, which will make promising targets for future mutagenesis studies were also identified. Finally, the conformational changes in 20S as induced by the transition of NSF from ATP to ADP state were modeled, and a concerted untwisting motion of SNARE/SNAPs and a sideway flip of two amino‐terminal domains were observed. In sum, the findings have offered new structural and dynamic details relevant to the SNARE disassembly mechanism, and will guide future functional studies of the SNARE recycling machinery. Proteins 2016; 84:1055–1066. © 2016 Wiley Periodicals, Inc.     

Remote Homology between Munc13 MUN Domain and Vesicle Tethering Complexes

Most core components of the neurotransmitter release machinery have homologues in other types of intracellular membrane traffic, likely underlying a universal mechanism of intracellular membrane fusion. However, no clear similarity between Munc13s and protein families generally involved in membrane traffic has been reported, despite the essential nature of Munc13s for neurotransmitter release. This crucial function was ascribed to a minimal Munc13 region called the MUN domain, which likely participates in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex (SNARE) assembly and is also found in Ca²⁺-dependent activator protein for secretion. We have now used comparative sequence and structural analyses to study the structure and evolutionary origin of the MUN domain. We found weak yet significant sequence similarities between the MUN domain and a set of protein subunits from several related vesicle tethering complexes, such as Sec6 from the exocyst complex and Vps53 from the Golgi-associated retrograde protein complex. Such an evolutionary relationship allows structure prediction of the MUN domain and suggests functional similarities between MUN domain-containing proteins and multisubunit tethering complexes such as exocyst, conserved oligomeric Golgi complex, Golgi-associated retrograde protein complex, and Dsl1p. These findings further unify the mechanism of neurotransmitter release with those of other types of intracellular membrane traffic and, in turn, support a role for tethering complexes in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex assembly.     

Accessory α-Helix of Complexin I Can Displace VAMP2 Locally in the Complexin-SNARE Quaternary Complex

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory α-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory α-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory α-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.     

Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function.     

NSF-fusion and beyond

NSF (N-ethylmaleimide-sensitive fusion protein) was the first protein to be isolated as a crucial factor in intracellular membrane-fusion events, such as the fusion of synaptic vesicles with the presynaptic membrane during neurotransmission. Although the activation of membrane SNARE proteins for subsequent fusion is clearly a primary role of NSF, recent studies have provided surprising evidence that NSF also interacts with glutamate receptors at the postsynaptic membrane in a way that does not seem to involve SNAREs. These results suggest that NSF might act as a molecular chaperone not only on SNAREs but also on other proteins.     

Arachidonic acid potentiates exocytosis and allows neuronal SNARE complex to interact with Munc18a

Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the neuronal plasma membrane. Recent genetic studies have highlighted the critical role played by polyunsaturated fatty acids in neurotransmission, however, there is little information available about which fatty acids act on exocytosis and, more importantly, by what mechanism. We have used permeabilized chromaffin cells to screen various fatty acids of the n-3 and n-6 series for their acute effects on exocytosis. We have demonstrated that an n-6 series polyunsaturated fatty acid, arachidonic acid, potentiates secretion from intact neurosecretory cells regardless of the secretagogue used. We have shown that arachidonic acid dose dependently increases soluble NSF attachment protein receptor complex formation in chromaffin cells and bovine cortical brain extracts and that a non-hydrolysable analogue of arachidonic acid causes a similar increase in SNARE complex formation. This prompted us to examine the effect of arachidonic acid on SNARE protein interactions with Munc18a, a protein known to prevent Syntaxin1a engagement into the SNARE complex in vitro. In the presence of arachidonic acid, we show that Munc18a can interact with the neuronal SNARE complex in a dose-dependent manner. We further demonstrate that arachidonic acid directly interacts with Syntaxin1a.     

Multiple roles of the vesicular-SNARE TI-VAMP in post-Golgi and endosomal trafficking

SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are the core machinery of membrane fusion. Vesicular SNAREs (v-SNAREs) interact with their target SNAREs (t-SNAREs) to form SNARE complexes which mediate membrane fusion. Here we review the basic properties and functions of the v-SNARE TI-VAMP/VAMP7 (Tetanus neurotoxin insensitive-vesicle associated membrane protein). TI-VAMP interacts with its t-SNARE partners, particularly plasmalemmal syntaxins, to mediate membrane fusion and with several regulatory proteins especially via its amino-terminal regulatory Longin domain. Partners include AP-3, Hrb/(Human immunodeficiency virus Rev binding) protein, and Varp (Vps9 domain and ankyrin repeats containing protein) and regulate TI-VAMP’s function and targeting. TI-VAMP is involved both in secretory and endocytic pathways which mediate neurite outgrowth and synaptic transmission, plasma membrane remodeling and lysosomal secretion.     

Cellular levels of the syntaxin Tlg2p are regulated by a single mode of binding to Vps45p

Sec1p/Munc18 (SM) proteins play a key role in the regulation of soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptor (SNARE)-mediated intracellular membrane trafficking events in all eukaryotic cells. Understanding the molecular mechanisms by which SM proteins function has not been straight forward as SM proteins bind to their cognate SNARE proteins by at least two distinct mechanisms, suggesting that they provide more than one function. We have previously characterised two binding modes used by the yeast SM protein Vps45p to interact with its SNARE proteins. In one of these modes, the N terminus of the syntaxin Tlg2p inserts into a hydrophobic pocket in the SM protein. We now report that disruption of this high-affinity binding between Vps45p and Tlg2p leads to downregulation of Tlg2p, and propose that this pocket-mode of binding of SM proteins to their cognate syntaxins serves to regulate cellular levels of the syntaxin.     

Multiple binding proteins suggest diverse functions for the N-ethylmaleimide sensitive factor

The hexameric ATPase, N-ethylmaleimide sensitive factor (NSF), is essential to vesicular transport and membrane fusion because it affects the conformations and associations of the soluble NSF attachment protein receptor (SNARE) proteins. NSF binds SNAREs through adaptors called soluble NSF attachment proteins (alpha- or beta-SNAP) and disassembles SNARE complexes to recycle the monomers. NSF contains three domains, two nucleotide-binding domains (NSF-D1 and -D2) and an amino terminal domain (NSF-N) that is required for SNAP-SNARE complex binding. Mutagenesis studies indicate that a cleft between the two sub-domains of NSF-N is critical for binding. The structural conservation of N domains in NSF, p97/VCP, and VAT suggests that a similar type of binding site could mediate substrate recognition by other AAA proteins. In addition to SNAP-SNARE complexes, NSF also binds other proteins and protein complexes such as AMPA receptor subunits (GluR2), beta2-adrenergic receptor, beta-Arrestin1, GATE-16, LMA1, rabs, and rab-containing complexes. The potential for these interactions indicates a broader role for NSF in the assembly/disassembly cycles of several cellular complexes and suggests that NSF may have specific regulatory effects on the functions of the proteins involved in these complexes. The structural requirements for these interactions and their physiological significance will be discussed.     

NSF and p97/VCP: similar at first, different at last

N-Ethylmaleimide sensitive factor (NSF) and p97/valosin-containing protein (VCP) are distantly related members of the ATPases associated with a variety of cellular activities (AAA) family of proteins. While both proteins have been implied in cellular morphology changes involving membrane compartments or vesicles, more recent evidence seems to imply that NSF is primarily involved in the soluble NSF attachment receptor (SNARE)-mediated vesicle fusion by disassembling the SNARE complex whereas p97/VCP is primarily involved in the extraction of membrane proteins. These functional differences are now corroborated by major structural differences based on recent crystallographic and cryo-electron microscopy studies. This review discusses these recent findings.     

An activated Q‐SNARE/SM protein complex as a possible intermediate in SNARE assembly

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18‐1 as a possible acceptor complex for the R‐SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18‐1 remains tethered by the N‐terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1‐65) was able to bind to the syntaxin1:SNAP25:Munc18‐1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R‐SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly.     

Kinesin-1 plays a role in transport of SNAP-25 to the plasma membrane

The cellular molecular motor kinesin-1 mediates the microtubule-dependent transport of a range of cargo. We have previously identified an interaction between the cargo-binding domain of kinesin-1 heavy chain KIF5B and the membrane-associated SNARE proteins SNAP-25 and SNAP-23. In this study we further defined the minimal SNAP-25 binding domain in KIF5B to residues 874-894. Overexpression of a fragment of KIF5B (residues 594-910) resulted in significant colocalization with SNAP-25 with resulting blockage of the trafficking of SNAP-25 to the periphery of cells. This indicates that kinesin-1 facilitates the transport of SNAP-25 containing vesicles as a prerequisite to SNAP-25 driven membrane fusion events.     

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.     

Membrane tethering and fusion in the secretory and endocytic pathways

Studies of intracellular trafficking over the past decade or so have led to striking advances in our understanding of the molecular processes by which transport intermediates dock and fuse. SNARE proteins play a central role, assembling into complexes that bridge membranes and may catalyze membrane fusion directly. In general, different SNARE proteins operate in different intracellular trafficking pathways, so recent reports that SNARE assembly in vitro is promiscuous have come as something of a surprise. We propose a model in which proper SNARE assembly is under kinetic control, orchestrated by members of the Sec1 protein family, small GTP-binding Rab proteins, and a diverse assortment of tethering proteins.     

Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation

Adenosine A_2A receptor (A_2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A_2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A_2AR expression are still obscure. Here, we demonstrated that 2 weeks of SAM treatment produced a significant reduction in the rat striatal A_2AR messenger RNA (mRNA) and protein content as well as A_2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5′UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A_2AR expression could be established. Overall, we concluded that striatal A_2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A_2AR expression.