Structural insights into the molecular mechanism of Ca(2+)-dependent exocytosis

The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Recent structures of the SNARE complex, synaptotagmin III, nSec1, domains of NSF and its adaptor SNAP, along with Rab3 and some of its effectors, provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models. Ultimately, this knowledge of the structures of higher-order complexes and their dynamic behavior will allow us to obtain a full understanding of the vesicle fusion protein machinery.     

Studies on the mechanism of membrane fusion: evidence for an intermembrane Ca2+-phospholipid complex, synergism with Mg2+, and inhibition by spectrin.

The interaction of Ca2+ and Mg2+ with phosphatidylserine (PS) vesicles in 0.1 M NaCl aqueous solution was studied by equilibrium dialysis binding, X-ray diffraction, batch microcalorimetry, kinetics of cation-induced vesicle aggregation, release of vesicle contents, and fusion. Addition of either cation causes aggregation of PS vesicles and produces complexes with similar stoichiometry (1:2 cation/PS) at saturating concentrations, although the details of the interactions and the resulting complexes are quite different. Addition of Ca2+ to PS vesicles at T greater than or equal to 25 degrees C induces the formation of an "anhydrous" complex of closely apposed membranes with highly ordered crystalline acyl chains and a very high transition temperature (Tc greater than 100 degrees C). The formation of this complex is accompanied by a release of heat (5.5 kcal/mol), rapid release of vesicle contents, and fusion of the vesicles into larger membranous structures. By contrast, addition of Mg2+ produces a complex with PS which is much more hydrated, has no crystallization of the acyl chains at T greater than or equal to 20 degrees C, and has comparatively little fusion. Studies with both Ca2+ and Mg2+ added simultaneously indicate that there is a synergistic effect between the two cations, which results in an enhancement of the ability of Ca2+ to form its specific complex with PS at lower concentrations. The presence of the erythrocyte protein "spectrin" inhibits this synergism and interferes with the formation of the specific PS/Ca complex. It also inhibits the fusion of PS vesicles. It is proposed that the unique PS/Ca complex, which involves close apposition of vesicle membranes, is an intermembrane "trans" complex. We further propose that such a complex is a key step for the resultant phase transition and fusion of PS vesicles. By contrast, the PS/Mg complex is proposed to be a "cis" complex with respect to each membrane. The results are discussed in terms of the mechanism of membrane fusion.     

The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila and C. elegans

Advances in the study of Drosophila melanogaster and Caenorhabditis elegans have provided key insights into the processes of neurotransmission and neuromodulation. Work in the past year has revealed that Unc-13 and Rab3a-interacting molecule regulate the conformational state of syntaxin to prime synaptic vesicle fusion. Analyses of synaptotagmin support its role as a putative calcium sensor triggering vesicular fusion and highlight the possible role of SNARE complex oligomerization in the fusion mechanism. Characterization of endophilin mutants demonstrates that kiss-and-run endocytosis is a major component of synaptic vesicle recycling. In neuromodulation, dcaps mutants provide the first genetic insight into possible roles of the CAPS protein in mediating dense core vesicle fusion and modulating synaptic vesicle fusion.     

Multi-step formation of a hemifusion diaphragm for vesicle fusion revealed by all-atom molecular dynamics simulations

Membrane fusion is essential for intracellular trafficking and virus infection, but the molecular mechanisms underlying the fusion process remain poorly understood. In this study, we employed all-atom molecular dynamics simulations to investigate the membrane fusion mechanism using vesicle models which were pre-bound by inter-vesicle Ca^2 +-lipid clusters to approximate Ca^2 +-catalyzed fusion. Our results show that the formation of the hemifusion diaphragm for vesicle fusion is a multi-step event. This result contrasts with the assumptions made in most continuum models. The neighboring hemifused states are separated by an energy barrier on the energy landscape. The hemifusion diaphragm is much thinner than the planar lipid bilayers. The thinning of the hemifusion diaphragm during its formation results in the opening of a fusion pore for vesicle fusion. This work provides new insights into the formation of the hemifusion diaphragm and thus increases understanding of the molecular mechanism of membrane fusion. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-α-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-α-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.     

Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined. In this report, we employ fluorescence resonance energy transfer technique to study the formation of SNARE complexes. Donor/acceptor pair variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) are fused with the N termini of SNAP-25 and synaptobrevin, respectively. In vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonance energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Under the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- synaptobrevin have no FRET. Adenovirus-mediated gene transfer is used to express the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular structures in PC12 cells co-expressing CFP-SNAP-25 and YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with sites presumed to be synaptic junctions. Neurosecretion in PC12 cells initiated by KCl depolarization leads to an increase in the extent of FRET. These results demonstrate that significant amounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/priming stage. Moreover, it demonstrates that FRET can be used as an effective tool for investigating dynamic SNARE interactions during synaptic vesicle fusion.     

Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis

Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.     

An Electrostatically Preferred Lateral Orientation of SNARE Complex Suggests Novel Mechanisms for Driving Membrane Fusion

Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins.     

Kiss-and-run, collapse and 'readily retrievable' vesicles

Two models of synaptic vesicle recycling have been intensely debated for decades: kiss‐and‐run, in which the vesicle opens and closes transiently, presumably through a small fusion pore, and full fusion, in which the vesicle collapses into the plasma membrane and is retrieved by clathrin‐coat‐dependent processes. Conceptually, it seems that kiss‐and‐run would be faster and would retrieve vesicles with greater fidelity. Is this the case? This review discusses recent evidence for both models. We conclude that both mechanisms allow for high fidelity of vesicle recycling. Also, the presence in the plasma membrane of a depot of previously fused vesicles that are already interacting with the endocytotic machinery (the ‘readily retrievable’ vesicles) allows full fusion to trigger quite fast endocytosis, further blurring the efficiency differences between the two models.     

Persistent fusion pores but transient fusion in alveolar type II cells

The release of vesicle contents following exocytotic fusion is limited by various factors including the size of the fusion pore. Fusion pores are channel-like, narrow structures after formation and proceed through semi-stable states ('fusion pore flickering'), unless they fully expand (full fusion) or close again (transient fusion). Partial release of vesicle contents may occur during transient fusion, which was described to last between milliseconds and seconds, depending on the size of the vesicle. We studied fusion pores in a slow-secreting lung epithelial cell (type II cell) using fluorescence staining of vesicle contents (surfactant) and fluorescence recovery after photobleaching (FRAP). Surfactant is a lipidic material, which is secreted into the alveolar lumen to reduce the surface tension in the lung. We found release of surfactant to be a slow process, which can last for hours. Accordingly, fusion pores in these cells are stable structures, which appear to be a barrier for release. FRAP measurements suggest that transient fusions occasionally take place in these long-lasting fusion pores, resulting in partial release of surfactant into the extracellular space. These data suggest that postfusion mechanisms may regulate the amount of secreted surfactant.     

Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion

In yeast, the Class C Vps protein complex (C-Vps complex), composed of Vps11, Vps16, Vps18, and Vps33, functions in Golgi-to-vacuole protein transport. In this study, we characterized and purified this complex and identified its interaction with the syntaxin homolog Vam3. Vam3 pairs with the SNAP-25 homolog Vam7 and VAMP homolog Vti1 to form SNARE complexes during vesicle docking/fusion with the vacuole. The C-Vps complex does not bind to Vam3-Vti1-Vam7 paired SNARE complexes but instead binds to unpaired Vam3. Antibodies to a component of this complex inhibited in vitro vacuole-to-vacuole fusion. Furthermore, temperature-conditional mutations in the Class C VPS genes destabilized Vam3-Vti1-Vam7 pairing. Therefore, we propose that the C-Vps complex associates with unpaired (activated) Vam3 to mediate the assembly of trans-SNARE complexes during both vesicle docking/fusion and vacuole-to-vacuole fusion.     

Complexins regulate a late step in Ca2+-dependent neurotransmitter release

Synaptic vesicle fusion at synapses is triggered by increases in cytosolic Ca2+ levels. However, the identity of the Ca2+ sensor and the transduction mechanism of the Ca2+ trigger are unknown. We show that Complexins, stoichiometric components of the exocytotic core complex, are important regulators of transmitter release at a step immediately preceding vesicle fusion. Neurons lacking Complexins show a dramatically reduced transmitter release efficiency due to decreased Ca2+ sensitivity of the synaptic secretion process. Analyses of mutant neurons demonstrate that Complexins are acting at or following the Ca2+-triggering step of fast synchronous transmitter release by regulating the exocytotic Ca2+ sensor, its interaction with the core complex fusion machinery, or the efficiency of the fusion apparatus itself.     

Calcium drives fusion of SNARE-apposed bilayers

N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) has been proposed to play a critical role in the membrane fusion process. The SNARE complex was suggested to be the minimal fusion machinery. However, there is mounting evidence for a major role of calcium in membrane fusion. Hence, the role of calcium in SNARE-induced membrane fusion was the focus of this study. It revealed that recombinant v-SNARE and t-SNARE, reconstituted into separate liposomes, interact to bring lipid vesicles into close proximity, enabling calcium to drive fusion of opposing bilayers. Exposure to calcium triggered vesicle fusion at both, high potency and efficacy. The half-time for calcium-induced fusion of SNARE-reconstituted vesicles was determined to be approximately 10 s, which is two orders of magnitude faster than in its absence. Calcium acts downstream of SNAREs, since the presence of SNAREs in bilayers increases the potency of calcium-induced vesicle fusion, without significantly influencing its efficacy. Hence, this study suggests that in the physiological state in cells, both SNAREs and calcium operate as the minimal fusion machinery.     

The Mechanism of a Nuclear Pore Assembly: A Molecular Biophysics View

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.     

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment.     

Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells

Secretion and membrane fusion are fundamental cellular processes involved in the physiology of health and disease. Studies within the past decade reveal the molecular mechanism of secretion and membrane fusion in cells. Studies reveal that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized plasma membrane structures. Swelling of secretory vesicles result in a build-up of intravesicular pressure, which allows expulsion of vesicular contents. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution, are discussed. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, have been resolved. With these findings a new understanding of cell secretion has emerged and confirmed by a number of laboratories.     

Retrograde vesicle transport in the Golgi

The Golgi apparatus is the central sorting and biosynthesis hub of the secretory pathway, and uses vesicle transport for the recycling of its resident enzymes. This system must operate with high fidelity and efficiency for the correct modification of secretory glycoconjugates. In this review, we discuss recent advances on how coats, tethers, Rabs and SNAREs cooperate at the Golgi to achieve vesicle transport. We cover the well understood vesicle formation process orchestrated by the COPI coat, and the comprehensively documented fusion process governed by a set of Golgi localised SNAREs. Much less clear are the steps in-between formation and fusion of vesicles, and we therefore provide a much needed update of the latest findings about vesicle tethering. The interplay between Rab GTPases, golgin family coiled-coil tethers and the conserved oligomeric Golgi (COG) complex at the Golgi are thoroughly evaluated.     

Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic,but neurotransmitter release persists

The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. We have investigated exocyst function in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, we found impairments in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5 differentiates between two forms of vesicle trafficking: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways.     

Major antiparallel and minor parallel β sheet populations detected in the membrane-associated human immunodeficiency virus fusion peptide

The HIV gp41 protein catalyzes fusion between viral and host cell membranes, and its apolar N-terminal region or "fusion peptide" binds to the host cell membrane and plays a key role in fusion. "HFP" is a construct containing the fusion peptide sequence, induces membrane vesicle fusion, and is an important fusion model system. Earlier solid-state nuclear magnetic resonance (SSNMR) studies showed that when HFP is associated with membranes with ～30 mol % cholesterol, the first 16 residues have predominant β strand secondary structure and a fraction of the strands form antiparallel β sheet structure with residue 16→1/1→16 or 17→1/1→17 registries for adjacent strands. In some contrast, other SSNMR and infrared studies have been interpreted to support a large fraction of an approximately in-register parallel registry of adjacent strands. However, the samples had extensive isotopic labeling, and other structural models were also consistent with the data. This SSNMR study uses sparse labeling schemes that reduce ambiguity in the determination of the fraction of HFP molecules with parallel β registry. Quantitative analysis of the data shows that the parallel fraction is at most 0.15 with a much greater fraction of antiparallel 16→1/1→16 and 17→1/1→17 registries. These data strongly support a model of HFP-induced vesicle fusion caused by antiparallel rather than parallel registries and provide insight into the arrangement of gp41 molecules during HIV-host cell fusion. This study is an example of quantitative determination of a complex structural distribution by SSNMR, including experimentally validated inclusion of natural abundance contributions to the SSNMR data.