PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

Structural Mechanism of Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Assembly in Lipid Bilayers Revealed by Solid-State NMR.

Synaptic vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including synaptobrevin-2 (Syb-2), syntaxin-1 (Syx-1), and SNAP-25. However, it remains controversial whether the formation of thoroughly contacted α-helical bundle from the SNARE motifs to the end of the transmembrane domains (TMDs) is necessary for SNARE-mediated membrane fusion. In this study, we characterized the conformation of Syb-2 in different assembly states using a combination of dipolar- and scalar-based solid-state NMR experiments in lipid bilayers. Our spectral analysis revealed a highly dynamic nature of the Syb-2 TMD with considerable α-helical contents. Chemical shift perturbation and mutational analysis indicated that the coupling between Syb-2 and Syx-1 TMDs mediated by residue Gly-100 of Syb-2 together with high mobility of the C-terminal segment of Syb-2 TMD are required for inner membrane merger. Our results provide new insights into the role of the Syb-2 TMD in driving membrane fusion, which improves the current understanding of the structural mechanism of SNARE complex assembly. This study highlights the significance of membrane environments in elucidating the mechanism of membrane proteins.

SNARE mimic peptide triggered membrane fusion kinetics revealed using single particle techniques.

Membrane fusion is an essential part of the proper functioning of life. As such it is not only important that organisms carefully regulate the process, but also that it is well understood. One way to facilitate and study membrane fusion is to use artificial, minimalist, fusion peptides. In this study the efficiency and kinetics of two fusion peptides, denoted CPE and CPK, were studied using single-particle TIRF microscopy. CPE and CPK are helical peptides which interact with each other, forming a coiled-coil motif. The peptides can be inserted into a lipid membrane using a lipid anchor, and if these peptides are anchored in opposing lipid membranes, then the coiled-coil interaction can provide the mechanical force necessary to overcome the energy barrier to initiate fusion, much in the same way the SNARE complex does. In this study we find that the fusogenic facilitation of CPE and CPK in liposomes is, at least partially, dependent on the size of the particle. In addition, under certain fusogenic conditions such as when using small liposomes of ∼60 nm in diameter, CPK alone is enough to facilitate membrane fusion in both bulk and single-particle studies. We show this using bulk lipid mixing assays utilizing FRET and single-particle TIRF, making use of dequenching fluorophores to indicate fusion. This provides us with new insights into the mechanisms of peptide-mediated membrane fusion and illuminates both challenges as well as opportunities when designing drug delivery systems.

Complexin-1 regulated assembly of single neuronal SNARE complex revealed by single-molecule optical tweezers.

The dynamic assembly of the Synaptic-soluble N-ethylmaleimide-sensitive factor Attachment REceptor (SNARE) complex is crucial to understand membrane fusion. Traditional ensemble study meets the challenge to dissect the dynamic assembly of the protein complex. Here, we apply minute force on a tethered protein complex through dual-trap optical tweezers and study the folding dynamics of SNARE complex under mechanical force regulated by complexin-1 (CpxI). We reconstruct the clamp and facilitate functions of CpxI in vitro and identify different interplay mechanism of CpxI fragment binding on the SNARE complex. Specially, while the N-terminal domain (NTD) plays a dominant role of the facilitate function, CTD is mainly related to clamping. And the mixture of 1-83aa and CTD of CpxI can efficiently reconstitute the inhibitory signal identical to that the full-length CpxI functions. Our observation identifies the important chaperone role of the CpxI molecule in the dynamic assembly of SNARE complex under mechanical tension, and elucidates the specific function of each fragment of CpxI molecules in the chaperone process.

Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy.

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.

Energetics, kinetics, and pathways of SNARE assembly in membrane fusion.

Fusion of transmitter-containing vesicles with plasma membranes at the synaptic and neuromuscular junctions mediates neurotransmission and muscle contractions, respectively, thereby underlying all thoughts and actions. The fusion process is driven by the coupled folding and assembly of three synaptic SNARE proteins--syntaxin-1 and SNAP-25 on the target plasma membrane (t-SNAREs) and VAMP2 on the vesicular membrane (v-SNARE) into a four-helix bundle. Their assembly is chaperoned by Munc18-1 and many other proteins to achieve the speed and accuracy required for neurotransmission. However, the physiological pathway of SNARE assembly and its coupling to membrane fusion remains unclear. Here, we review recent progress in understanding SNARE assembly and membrane fusion, with a focus on results obtained by single-molecule manipulation approaches and electric recordings of single fusion pores. We describe two pathways of synaptic SNARE assembly, their associated intermediates, energetics, and kinetics. Assembly of the three SNAREs in vitro begins with the formation of a t-SNARE binary complex, on which VAMP2 folds in a stepwise zipper-like fashion. Munc18-1 significantly alters the SNARE assembly pathway: syntaxin-1 and VAMP2 first bind on the surface of Munc18-1 to form a template complex, with which SNAP-25 associates to conclude SNARE assembly and displace Munc18-1. During membrane fusion, multiple trans-SNARE complexes cooperate to open a dynamic fusion pore in a manner dependent upon their copy number and zippering states. Together, these results demonstrate that stepwise and cooperative SNARE assembly drive stagewise membrane fusion.

Molecular Mechanisms Underlying Neurotransmitter Release.

Major recent advances and previous data have led to a plausible model of how key proteins mediate neurotransmitter release. In this model, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin form tight complexes that bring the membranes together and are crucial for membrane fusion. NSF and SNAPs disassemble SNARE complexes and ensure that fusion occurs through an exquisitely regulated pathway that starts with Munc18-1 bound to a closed conformation of syntaxin-1. Munc18-1 also binds to synaptobrevin, forming a template to assemble the SNARE complex when Munc13-1 opens syntaxin-1 while bridging the vesicle and plasma membranes. Synaptotagmin-1 and complexin bind to partially assembled SNARE complexes, likely stabilizing them and preventing fusion until Ca2+ binding to synaptotagmin-1 causes dissociation from the SNARE complex and induces interactions with phospholipids that help trigger release. Although fundamental questions remain about the mechanism of membrane fusion, these advances provide a framework to investigate the mechanisms underlying presynaptic plasticity.

The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines.

ATPases associated with diverse cellular activities (AAA+ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+ domain responsible for a diverse range of cellular activities. Typically, AAA+ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+ fold is presented. Next, the seven different clades of AAA+ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+ proteins, NSF and its close relative p97, are reviewed in detail.

α-Synuclein kinetically regulates the nascent fusion pore dynamics.

α-Synuclein (α-synFL) is central to the pathogenesis of Parkinson's disease (PD), in which its nonfunctional oligomers accumulate and result in abnormal neurotransmission. The normal physiological function of this intrinsically disordered protein is still unclear. Although several previous studies demonstrated α-synFL's role in various membrane fusion steps, they produced conflicting outcomes regarding vesicular secretion. Here, we assess α-synFL's role in directly regulating individual exocytotic release events. We studied the micromillisecond dynamics of single recombinant fusion pores, the crucial kinetic intermediate of membrane fusion that tightly regulates the vesicular secretion in different cell types. α-SynFL accessed v-SNARE within the trans-SNARE complex to form an inhibitory complex. This activity was dependent on negatively charged phospholipids and resulted in decreased open probability of individual pores. The number of trans-SNARE complexes influenced α-synFL's inhibitory action. Regulatory factors that arrest SNARE complexes in different assembly states differentially modulate α-synFL's ability to alter fusion pore dynamics. α-SynFL regulates pore properties in the presence of Munc13-1 and Munc18, which stimulate α-SNAP/NSF-resistant SNARE complex formation. In the presence of synaptotagmin1(syt1), α-synFL contributes with apo-syt1 to act as a membrane fusion clamp, whereas Ca2+•syt1 triggered α-synFL-resistant SNARE complex formation that rendered α-synFL inactive in modulating pore properties. This study reveals a key role of α-synFL in controlling vesicular secretion.

En route to dynamic life processes by SNARE-mediated fusion of polymer and hybrid membranes.

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.

Tight docking of membranes before fusion represents a metastable state with unique properties.

Membrane fusion is fundamental to biological processes as diverse as membrane trafficking or viral infection. Proteins catalyzing membrane fusion need to overcome energy barriers to induce intermediate steps in which the integrity of bilayers is lost. Here, we investigate the structural features of tightly docked intermediates preceding hemifusion. Using lipid vesicles in which progression to hemifusion is arrested, we show that the metastable intermediate does not require but is enhanced by divalent cations and is characterized by the absence of proteins and local membrane thickening. Molecular dynamics simulations reveal that thickening is due to profound lipid rearrangements induced by dehydration of the membrane surface.

Chaperoning SNARE Folding and Assembly.

SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.

Membrane fusion mediated by peptidic SNARE protein analogues: Evaluation of FRET-based bulk leaflet mixing assays.

Peptide-mediated membrane fusion is frequently studied with in vitro bulk leaflet mixing assays based on Förster resonance energy transfer (FRET). In these, customized liposomes with fusogenic peptides are equipped with lipids which are labeled with fluorophores that form a FRET pair. Since FRET is dependent on distance and membrane fusion comes along with lipid mixing, the assays allow for conclusions on the membrane fusion process. The experimental outcome of these assays, however, greatly depends on the applied parameters. In the present study, the influence of the peptides, the size of liposomes, their lipid composition and the liposome stoichiometry on the fusogenicity of liposomes are evaluated. As fusogenic peptides, soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein analogues featuring artificial recognition units attached to the native SNARE transmembrane domains are used. The work shows that it is important to control these parameters in order to be able to properly investigate the fusion process and to prevent undesired effects of aggregation.

Analysis of the SNARE Stx8 recycling reveals that the retromer-sorting motif has undergone evolutionary divergence.

Fsv1/Stx8 is a Schizosaccharomyces pombe protein similar to mammalian syntaxin 8. stx8Δ cells are sensitive to salts, and the prevacuolar endosome (PVE) is altered in stx8Δ cells. These defects depend on the SNARE domain, data that confirm the conserved function of syntaxin8 and Stx8 in vesicle fusion at the PVE. Stx8 localizes at the trans-Golgi network (TGN) and the prevacuolar endosome (PVE), and its recycling depends on the retromer component Vps35, and on the sorting nexins Vps5, Vps17, and Snx3. Several experimental approaches demonstrate that Stx8 is a cargo of the Snx3-retromer. Using extensive truncation and alanine scanning mutagenesis, we identified the Stx8 sorting signal. This signal is an IEMeaM sequence that is located in an unstructured protein region, must be distant from the transmembrane (TM) helix, and where the 133I, 134E, 135M, and 138M residues are all essential for recycling. This sorting motif is different from those described for most retromer cargoes, which include aromatic residues, and resembles the sorting motif of mammalian polycystin-2 (PC2). Comparison of Stx8 and PC2 motifs leads to an IEMxx(I/M) consensus. Computer-assisted screening for this and for a loose Ψ(E/D)ΨXXΨ motif (where Ψ is a hydrophobic residue with large aliphatic chain) shows that syntaxin 8 and PC2 homologues from other organisms bear variation of this motif. The phylogeny of the Stx8 sorting motifs from the Schizosaccharomyces species shows that their divergence is similar to that of the genus, showing that they have undergone evolutionary divergence. A preliminary analysis of the motifs in syntaxin 8 and PC2 sequences from various organisms suggests that they might have also undergone evolutionary divergence, what suggests that the presence of almost-identical motifs in Stx8 and PC2 might be a case of convergent evolution.

Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact.

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

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.

Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

SNAREs in Plant Biotic and Abiotic Stress Responses.

In eukaryotes, membraneous cellular compartmentation essentially requires vesicle trafficking for communications among distinct organelles. A donor organelle-generated vesicle releases its cargo into a target compartment by fusing two distinct vesicle and target membranes. Vesicle fusion, the final step of vesicle trafficking, is driven intrinsically by complex formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNAREs are well-conserved across eukaryotes, genomic studies revealed that plants have dramatically increased the number of SNARE genes than other eukaryotes. This increase is attributed to the sessile nature of plants, likely for more sensitive and harmonized responses to environmental stresses. In this review, we therefore try to summarize and discuss the current understanding of plant SNAREs function in responses to biotic and abiotic stresses.

Prototypic SNARE Proteins Are Encoded in the Genomes of Heimdallarchaeota, Potentially Bridging the Gap between the Prokaryotes and Eukaryotes.

A defining feature of eukaryotic cells is the presence of numerous membrane-bound organelles that subdivide the intracellular space into distinct compartments. How the eukaryotic cell acquired its internal complexity is still poorly understood. Material exchange among most organelles occurs via vesicles that bud off from a source and specifically fuse with a target compartment. Central players in the vesicle fusion process are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. These small tail-anchored (TA) membrane proteins zipper into elongated four-helix bundles that pull membranes together. SNARE proteins are highly conserved among eukaryotes but are thought to be absent in prokaryotes. Here, we identified SNARE-like factors in the genomes of uncultured organisms of Asgard archaea of the Heimdallarchaeota clade, which are thought to be the closest living relatives of eukaryotes. Biochemical experiments show that the archaeal SNARE-like proteins can interact with eukaryotic SNARE proteins. We did not detect SNAREs in α-proteobacteria, the closest relatives of mitochondria, but identified several genes encoding for SNARE proteins in γ-proteobacteria of the order Legionellales, pathogens that live inside eukaryotic cells. Very probably, their SNAREs stem from lateral gene transfer from eukaryotes. Together, this suggests that the diverse set of eukaryotic SNAREs evolved from an archaeal precursor. However, whether Heimdallarchaeota actually have a simplified endomembrane system will only be seen when we succeed studying these organisms under the microscope.