Multiple intermediates in SNARE-induced membrane fusion

111

Membrane fusion entails organelle docking and subsequent mixing of membrane bilayers and luminal compartments. We now present an in vitro assay of fusion, using yeast vacuoles bearing domains of either Fos or Jun fused to complementary halves of ␤-lactamase. Upon fusion, these proteins associate to yield ␤-lactamase activity. This assay complements the standard fusion assay (activation of pro-Pho8p in protease-deficient vacuoles by proteases from pho8⌬ vacuoles). Both the ␤-lactamase and proPho8p activation assays of fusion show the same long kinetic delay between SNARE pairing and luminal compartment mixing. Lipidmixing occurs rapidly after SNARE pairing but well before aqueous compartment mixing. These results support a model in which SNARE pairing leads to rapid hemifusion, followed by slow further lipid rearrangement and aqueous compartment mixing. (diacylglycerol, phosphoinositides, and ergosterol) are concentrated at a ring-shaped microdomain, the ''vertex ring,'' surrounding the apposed membranes of docked vacuoles. During docking, the released Vam7p rebinds to the vacuole through its affinities for phosphatidylinositol 3-phosphate (1) and HOPS (2) and participates in the formation of trans-SNARE complexes between apposed vacuolar membranes. Bilayer fusion around the vertex ring permits lipid and aqueous compartment mixing. It has been proposed that membrane fusion may require a lipidic ''hemifusion'' intermediate, where the closely apposed membrane leaflets are fused and mix, whereas the distal leaflets and aqueous compartments remain distinct. Hemifusion has been directly observed with influenza HA (3-5), either wild-type or with a GPI-anchor in place of its transmembrane domain (TMD), in a minimal SNARE-driven liposome fusion system (6, 7) with low levels of wild-type SNARE proteins or with mutant SNARE proteins with partial TMDs that span only one bilayer leaflet (6). Hemifusion was also observed in cell-cell fusion mediated by GPI-anchored SNARE proteins expressed on the outer surface of the plasma membrane (8), suggesting that all SNARE-dependent fusion might proceed by means of hemifusion. A hemifusion intermediate was proposed for yeast vacuole fusion, based on the finding that GTP␥S allows lipid mixing while blocking the fusion-dependent activation of pro-Pho8p (9). The standard in vitro assay of vacuole fusion measures the proteolytic activation of the inactive proPho8p (alkaline phosphatase or ALP) by vacuolar proteases upon content mixing between two populations of vacuoles. One limitation of this assay is its sensitivity to reagents that inhibit the activation of ALP. We have therefore developed an in vitro assay of yeast vacuole fusion in which content mixing between two vacuole populations is measured by the reconstitution of active Escherichia coli TEM-1 ␤-lactamase from two complementary fragments of the enzyme, each contributed from distinct vacuole populations. This fusion assay, coupled with a lipid mixing assay, has revealed that docking and lipid mixing are completed long before l...

Section: Discussionmentioning

confidence: 71%

Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

Jun

Wickner

2007

111

“…For example, in the case of synaptic transmission, the reaction is tightly regulated by a number of proteins of which the SNAREs [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors] have been shown to play a crucial role (1)(2)(3). In vitro studies have demonstrated the sufficiency of SNAREs to mediate fusion when reconstituted into a number of model systems, including freestanding lipid vesicles (4-7), supported lipid bilayers (8)(9)(10), and tethered vesicles (11). Generally these studies suggest that SNAREs are able to bring two membranes into close apposition by forming a trans complex in a docking reaction which is highly energetically favorable (12).…”

mentioning

confidence: 99%

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Chan

Lengerich

Boxer

2009

278

287

Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5 -coupled conjugate and the other the 3 -coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.DNA machine ͉ synthetic biology F usion of lipid membranes plays a central role in many biological processes. For example, in the case of synaptic transmission, the reaction is tightly regulated by a number of proteins of which the SNAREs [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors] have been shown to play a crucial role (1-3). In vitro studies have demonstrated the sufficiency of SNAREs to mediate fusion when reconstituted into a number of model systems, including freestanding lipid vesicles (4-7), supported lipid bilayers (8-10), and tethered vesicles (11). Generally these studies suggest that SNAREs are able to bring two membranes into close apposition by forming a trans complex in a docking reaction which is highly energetically favorable (12). This is illustrated in Fig. 1A, which is based on the x-ray structures of the soluble domains of SNARE proteins in complex (13). Several steps along the mechanism to achieve membrane fusion following this basic docking step have been proposed, and some of these are illustrated in Fig. 2, which also serves to illustrate assays commonly used to assess their role. In this step-wise mechanism, the bilayers first dock, then adopt an intermediate, hemifused state in which the outer leaflets have merged, but the inner leaflets and contents remain distinct. A transition into full fusion then involves mixing of both leaflets and content exchange.Despite substantial research on the role of SNARE proteins in the fusion process, many questions remain regarding the physical mechanism. Studies using simpler model systems can provide insight into the fundamental mechanisms of the rearrangement of bilayers and lead to a better understanding of the biological process (14, 15). We have prepared a series of DNA-lipid conjugates in which DNA o...

“…When tethering ligands such as biotin (21)(22)(23)(24) or a DNA oligonucleotide (11,12,25) are displayed on the supported bilayer, then individual intact vesicles displaying the complementary receptor can be held close to the surface where they can be visualized. Biotin-streptavidin tethers have been used extensively to isolate individual vesicles primarily in the context of single-molecule fluorescence measurements (21,24). In our experience, such tethered vesicles are relatively immobile on the surface, which is ideal for single-molecule measurements but less useful for probing interactions between tethered vesicles.…”

mentioning

confidence: 99%

Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers

Chan

Lenz²,

Boxer³

2007

Membrane-membrane recognition and binding are crucial in many biological processes. We report an approach to studying the dynamics of such reactions by using DNA-tethered vesicles as a general scaffold for displaying membrane components. This system was used to characterize the docking reaction between two populations of tethered vesicles that display complementary DNA. Deposition of vesicles onto a supported lipid bilayer was performed by using a microfluidic device to prevent mixing of the vesicles in bulk during sample preparation. Once tethered onto the surface, vesicles mixed via two-dimensional diffusion. DNA-mediated docking of two reacting vesicles results in their colocalization after collision and their subsequent tandem motion. Individual docking events and population kinetics were observed via epifluorescence microscopy. A lattice-diffusion simulation was implemented to extract from experimental data the probability, P dock, that a collision leads to docking. For individual vesicles displaying small numbers of docking DNA, P dock shows a first-order relationship with copy number as well as a strong dependence on the DNA sequence. Both trends are explained by a model that includes both tethered vesicle diffusion on the supported bilayer and docking DNA diffusion over each vesicle's surface. These results provide the basis for the application of tethered vesicles to study other membrane reactions including protein-mediated docking and fusion.