An InCytes from MBC Selection: Yeast Sec1p Functions before and after Vesicle Docking

Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.     

Formation and Turnover of NSF- and SNAP-containing “Fusion” Complexes Occur on Undocked, Clathrin-coated Vesicle–derived Membranes

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle–derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.     

Effects of cholesterol on the divalent cation-mediated interactions of vesicles containing amino and choline phospholipids

We have used assays of lipid probe mixing, contents mixing and contents leakage to monitor the divalent cation-mediated interactions between lipid vesicles containing phosphatidylserine (PS) as a minority component together with mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC) or sphingomyelin, and cholesterol in varying proportions. The initial rates of calcium- and magnesium-induced lipid probe quenching between vesicles, which reflect primarily the rates of vesicle aggregation, are strongly reduced as progressively higher proportions of PC or sphingomyelin are incorporated into PE/PS vesicles. The initial rates of divalent cation-induced contents mixing and contents leakage for PE/PS vesicles are also strongly reduced when choline phospholipids are incorporated into the vesicles in even low molar proportions. Sphingomyelin has a more potent inhibitory effect on these processes than does PC at an equal level in the vesicle membranes. The inclusion of cholesterol in these vesicles, at levels up to 1:2 moles sterol/mole phospholipid, has little effect on the rates of calcium- or magnesium-induced vesicle aggregation. However, cholesterol significantly enhances the initial rates of vesicle contents mixing and contents leakage in the presence of divalent cations when the vesicles contain choline as well as amino phospholipids. This effect is substantial only when the level of cholesterol exceeds the level of choline phospholipids in the vesicles. These results may have significance for the fusion of certain cellular membranes in mammalian cells, whose cytoplasmic faces have lipid compositions very similar to those of the vesicles examined in this study.     

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

The Ca²⁺-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.     

SNARE-driven, 25-millisecond vesicle fusion in vitro

Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate k(dock) and the unimolecular fusion rate k(fus). On low t-SNARE-density bilayers at 37 degrees C, docking is efficient: k(dock) = 2.2 x 10(7) M(-1) s(-1), approximately 40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (D(lipid) = 0.6 microm2 s(-1)); approximately 80% of the docked vesicles fuse promptly as a homogeneous subpopulation with k(fus) = 40 +/- 15 s(-1) (tau(fus) = 25 ms). This is 10(3)-10(4) times faster than previous in vitro fusion assays. Complete lipid mixing occurs in <15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca2+ is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains approximately 40 times slower than the fastest, submillisecond presynaptic vesicle population response.     

Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.

According to the soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements of SNARE interactions in planar supported membranes could potentially resolve some of the key questions regarding the mechanism of SNARE-mediated membrane fusion. As a first step toward this goal, recombinant syntaxin1A/SNAP25 (t-SNARE) was reconstituted into polymer-supported planar lipid bilayers. Reconstituted t-SNAREs in supported bilayers bound soluble green fluorescent protein/vesicle-associated membrane protein (v-SNARE), and the SNARE complexes could be specifically dissociated by NSF/alpha-SNAP in the presence of ATP. The physiological activities of SNARE complex formation were thus well reproduced in this reconstituted planar model membrane system. A large fraction (~75%) of the reconstituted t-SNARE was laterally mobile with a lateral diffusion coefficient of 7.5 x 10(-9) cm(2)/s in a phosphatidylcholine lipid background. Negatively charged lipids reduced the mobile fraction of the t-SNARE and the lipids themselves. Phosphatidylinositol-4,5-bisphosphate was more effective than phosphatidylserine in reducing the lateral mobility of the complexes. A model of how acidic lipid-SNARE interactions might alter lipid fluidity is discussed.     

Structural insights into the SNARE mechanism

Eukaryotic cells distribute materials among intracellular organelles and secrete into the extracellular space through cargo-loaded vesicles. A concluding step during vesicular transport is the fusion of a transport vesicle with a target membrane. SNARE proteins are essential for all vesicular fusion steps, thus they possibly comprise a conserved membrane fusion machinery. According to the "zipper" model, they assemble into stable membrane-bridging complexes that gradually bring membranes in juxtaposition. Hence, complex formation may provide the necessary energy for overcoming the repulsive forces between two membranes. During the last years, detailed structural and functional studies have extended the evidence that SNAREs are mostly in accord with the zipper model. Nevertheless, it remains unclear whether SNARE assembly between membranes directly leads to the merger of lipid bilayers.     

Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.     

Single SNARE-mediated vesicle fusion observed in vitro by polarized TIRFM

Single-vesicle fusion assays in vitro are useful tools for examining mechanisms of membrane fusion at the molecular level mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). This approach allows the experimentalist to define the lipid and protein composition of the two fusing membranes and perform experiments under highly controlled conditions. In previous experiments, in which we reconstituted a SNARE acceptor complex into supported membranes and observed the docking and fusion of fluorescently labeled synaptobrevin proteoliposomes by total internal reflection fluorescence microscopy with millisecond time resolution, we were able to determine the optimal number of SNARE complexes needed for fast fusion. Here, we utilize this assay in combination with polarized total internal reflection fluorescence microscopy to investigate topology changes that vesicles undergo after the onset of fusion. The theory that describes the fluorescence intensity during the transformation of a single vesicle from a spherical particle to a flat membrane patch is developed and confirmed by experiments with three different fluorescent probes. Our results show that on average, the fusing vesicles flatten and merge into the planar membrane within 8 ms after fusion starts.     

Syntaxin Is Efficiently Excluded from Sphingomyelin-enriched Domains in Supported Lipid Bilayers Containing Cholesterol

Formation of a trans-complex between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25 drives membrane fusion. The structure of the core SNARE complex has been studied extensively. Here we have used atomic force microscopy to study the behavior of recombinant syntaxin 1A both in detergent extracts and in a lipid environment. Full-length syntaxin in detergent extracts had a marked tendency to aggregate, which was countered by addition of munc-18. In contrast, syntaxin lacking its transmembrane region was predominantly monomeric. Syntaxin could be integrated into liposomes, which formed lipid bilayers when deposited on a mica support. Supported bilayers were decorated with lipid vesicles in the presence, but not the absence, of full-length syntaxin, indicating that formation of syntaxin complexes in trans could mediate vesicle docking. Syntaxin complexes remained at the sites of docking following detergent solubilization of the lipids. Raised lipid domains could be seen in bilayers containing sphingomyelin, and these domains were devoid of syntaxin and docked vesicles in the presence, but not the absence, of cholesterol. Our results demonstrate that syntaxin is excluded from sphingomyelin-enriched domains in a cholesterol-dependent manner.     

All three components of the neuronal SNARE complex contribute to secretory vesicle docking

Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs.     

Parameters affecting fusion between Sendai virus and liposomes. Role of viral proteins, liposome composition, and pH

A kinetic and quantitative characterization of the fusion process between Sendai virus and phospholipid vesicles is presented. Membrane fusion was monitored in a direct and continuous manner by employing an assay which relies on the relief of fluorescence self-quenching of the probe octadecylrhodamine B chloride which was located in the viral membrane. Viral fusion activity was strongly dependent on the vesicle lipid composition and was most efficient with vesicles solely consisting of acidic phospholipids, particularly cardiolipin (CL). This result implies that the fusion of viruses with liposomes does not display an absolute requirement for specific membrane receptors. Incorporation of phosphatidylcholine (PC), rather than phosphatidylethanolamine (PE), into CL bilayers strongly inhibited fusion, suggesting that repulsive hydration forces interfere with the close approach of viral and target membrane. Virus-liposome fusion products were capable of fusing with liposomes, but not with virus. In contrast to fusion with erythrocyte membranes, fusion between virus and acidic phospholipid vesicles was triggered immediately, did not strictly depend on viral protein conformation, and did not display a pH optimum around pH 7.5. On the other hand, with vesicles consisting of PC, PE, cholesterol, and the ganglioside GD1a, the virus resembled more closely the fusogenic properties that were seen with erythrocyte target membranes. Upon decreasing the pH below 5.0, the viral fusion activity increased dramatically. With acidic phospholipid vesicles, maximal activity was observed around pH 4.0, while with GD1a-containing zwitterionic vesicles the fusion activity continued to increase with decreasing pH down to values as low as 3.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION*

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075–11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.     

Importance of phosphatidylethanolamine for the interaction of apocytochrome c with model membranes containing phosphatidylserine

The effect of phosphatidylethanolamine (PE) on the binding of apocytochrome c to model membranes was examined. When 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of the standard vesicles composed of 80% of this lipid and 20% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) was gradually replaced with upward of 50% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), the binding increased appreciably. Ca(2+), causing the phase separation of PS, also brought about increased binding of apocytochrome c in the PC/PS system, underlining the importance of PS properties in membranes for the protein binding. The resonance energy transfer between Trp-59 in apocytochrome c and pyrene-PS incorporated into bilayers showed that the replacement of PC with PE increased the extent of apocytochrome c penetration into membranes by a PE concentration-dependent manner. However, in the absence of PS, PE had no apparent effect on these functions of apocytochrome c, suggesting that PE-induced change(s) of acidic membrane properties is important to the association of apocytochrome c with vesicles. From the observations that the excimer to monomer fluorescence ratio of pyrene-PS increased and the fluorescence of NBD-PS was quenched with increasing concentration of PE, it was deduced that PE caused PS-enriched domains in PC/PE/PS membranes. The colocalization of pyrene-PS with BODIPY-PS by PE further supported the possibility. We suggest that PE-induced formation of PS-enriched domains acts as binding sites for apocytochrome c in membranes.     

The curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, approximately 40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained approximately 20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets of the bilayer. The proportion of total PE residing in the outer leaflet was unaffected by changes in either the cholesterol or PE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate- and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions of palmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

The Curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content,and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm)radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, ~40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained ~20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets ofthe bilayer. The proportion oftotal PE residing in the outer leaflet was unaffected by changes in either the cholesterol orPE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions ofpalmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

Structure of membrane‐associated neuronal SNARE complex: implication in neurotransmitter release

To enable fusion between biological membranes, t‐SNAREs and v‐SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring‐complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t‐/v‐SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE‐ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40–50 nm in diameter membrane‐bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12–17 nm in diameter cup‐shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t‐SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t‐/v‐SNARE complex, 50 nm t‐and v‐SNARE proteoliposomes were mixed, allowing t‐SNARE‐vesicles to interact with v‐SNARE vesicles, followed by detergent solubilization and imaging of the resultant t‐/v‐SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6–7 nm membrane‐directed self‐assembled t‐/v‐SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3–4 nm base diameter, where 40–50 nm in diameter v‐SNARE‐associated synaptic vesicle transiently dock and fuse to release neurotransmitters.     

Lipidic antagonists to SNARE-mediated fusion

SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.     

Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.

ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy. COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p. LMA1 and Sec18p are required for vesicle fusion after Uso1p function. Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p. Once docked, however, vesicle fusion is no longer sensitive to GDI. In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding to membranes. To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed. These mutations do not interfere with Uso1p-mediated docking, but block membrane fusion. We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.     

神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.