SNAREs can promote complete fusion and hemifusion as alternative outcomes

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: 70%

mentioning

confidence: 99%

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

Jun

Wickner

2007

111

“…To test this, we developed a cell-cell fusion assay to detect lipid mixing (hemifusion) as well as content mixing (full fusion) based on a soluble NSF attachment receptor (SNARE) hemifusion assay reported previously (16). CHO cells lack the lipid ganglioside GM1 (16), so lipid mixing could be detected by using a TRITC-conjugated form of cholera toxin ␤-subunit (CTX) when CHO cells were fused to cells that express GM1.…”

Section: Results Gb Is Not Required For Hemifusionmentioning

confidence: 99%

Herpes simplex virus type 1 mediates fusion through a hemifusion intermediate by sequential activity of glycoproteins D, H, L, and B

Subramanian

Geraghty

2007

158

145

Virus-induced membrane fusion can be subdivided into three phases defined by studies of class I and class II fusion proteins. During Phase I, two membranes are brought into close apposition. Phase II marks the mixing of the outer membrane leaflets leading to formation of a hemifusion intermediate. A fusion pore stably forms and expands in Phase III, thereby completing the fusion process. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins to complete membrane fusion, but none has been defined as class I or II. Therefore, we investigated whether HSV-1-induced membrane fusion occurred following the same general phases as those described for class I and II proteins. In this study we demonstrate that glycoprotein D (gD) and the glycoprotein H and glycoprotein L complex (gHL) mediated lipid mixing indicative of hemifusion. However, content mixing and full fusion required glycoprotein B (gB) to be present along with gD and gHL. Our results indicate that, like class I and II fusion proteins, fusion mediated by HSV-1 glycoproteins occurred through a hemifusion intermediate. In addition, both gB and gHL are probably directly involved in the fusion process. From this, we propose a sequential model for fusion via HSV-1 glycoproteins whereby gD is required for Phase I, gHL is required for Phase II, and gB is required for Phase III. We further propose that glycoprotein H and gB are likely to function sequentially to promote membrane fusion in other herpesviruses such as Epstein-Barr virus and human herpesvirus 8.lipid transfer ͉ membrane fusion ͉ fluorescence microscopy S tudies using class I and class II fusion proteins have demonstrated that virus-induced membrane fusion can be subdivided into three phases (1, 2). Phase I involves bringing opposing membranes into close proximity through a viral glycoprotein binding a cellular receptor. Phase II involves the initiation of lipid mixing between the two apposed membranes and is completed when the outer membrane leaflets are mixed to form an intermediate called hemifusion. Phase III begins when the inner membrane leaflets are mixed and continues the pore formation and expansion. The completion of Phase III signifies the completion of the fusion process. The three phases of membrane fusion (close apposition, hemifusion, and complete fusion) are useful to characterize functions of viral glycoproteins in the fusion process (1, 3, 4). Fusion proteins that have Phase I function bring membranes in close apposition and ultimately result in the initiation of the fusion process. Fusion proteins that have Phase II function are capable of mixing outer membrane leaflets leading to hemifusion. Phase III fusion proteins are capable of forming and expanding a fusion pore. For many viruses, one or two fusion proteins can carry out all phases of membrane fusion. The fusion process has yet to be characterized for viruses that require more than two fusion glycoproteins, and a major issue is whether multiple glycoproteins mediate fusion through a hemifusion intermediate.Herpes simplex ...

“…Recent studies have suggested that this process proceeds through a hemifused state in which the outer membrane leaflets fuse first, followed by fusion of the inner membrane leaflets (10)(11)(12)(13). Formation of the hemifused state is characterized by significant physical constraints, because the outer leaflet needs to bend in a tight negative curve to preserve membrane integrity.…”

Section: Snap23 ͉ Syntaxin 4 ͉ Vamp2mentioning

confidence: 99%

“…It has been recently reported that SNARE-mediated membrane fusion occurs through a hemifusion intermediate state (10)(11)(12)(13). Therefore, we next determined the effect of different phospholipid compositions on the proportion of hemifused versus fused membrane states.…”

Section: The Hemifusion Step Is the Rate-limiting Factor For In Vitromentioning

confidence: 99%

Asymmetric phospholipid distribution drives in vitro reconstituted SNARE-dependent membrane fusion

Vicogne

Vollenweider

Smith

et al. 2006

103

Insulin-stimulated glucose uptake requires the fusion of GLUT4 transporter-containing vesicles with the plasma membrane, a process that depends on the SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment receptor) proteins VAMP2 (vesicleassociated membrane protein 2) and syntaxin 4 (Stx4)͞SNAP23 (soluble N-ethylmaleimide-sensitive fusion factor attachment protein 23). Efficient SNARE-dependent fusion has been shown in many settings in vivo to require the generation of both phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidic acid (PA). Addition of PA to Stx4͞SNAP23 vesicles markedly enhanced the fusion rate, whereas its addition to VAMP2 vesicles was inhibitory. In contrast, addition of PIP2 to Stx4͞SNAP23 vesicles inhibited the fusion reaction, and its addition to VAMP2 vesicles was stimulatory. The optimal distribution of phospholipids was found to trigger the progression from the hemifused state to full fusion. These findings reveal an unanticipated dependence of SNARE complex-mediated fusion on asymmetrically distributed acidic phospholipids and provide mechanistic insights into the roles of phospholipase D and PIP kinases in the late stages of regulated exocytosis.SNAP23 ͉ syntaxin 4 ͉ VAMP2