when the SNARE Vam7p was added in excess, which results in increased trans-SNARE complex formation, vacuole lysis increased (Vam7p has a PX domain for membrane association, but no transmembrane anchor). The Vam7p-induced lysis was concentration dependent and required full-length Vam7p ca- pable of SNARE pairing. Similarly, vacuoloes isolated from strains overexpressing all four vacuolar SNARE proteins were also prone to lysis. Vacuole lysis was blocked by antibodies that inhibit cis-SNARE disassembly, vacuole docking, and trans- SNARE pairing. Furthermore, vacuole lysis and vacuole fusion followed identical kinetics. Vacuole lysis by high SNARE activity compliments ear- lier observations regarding SNARE-containing liposome integ- rity after reconstitution of neuronal SNAREs (Dennison et al., 2006). Vesicles containing syntaxin at a high protein/lipid ratio exhibited increased contents leakage. Together, these studies suggest that although SNAREs are the minimal bilayer destabi- lization machinery, other factors assist in converting membrane destabilization to membrane fusion. An exciting explanation for SNARE-dependent vacuole lysis is that trans-SNARE pairs are balanced with regulatory proteins that govern membrane integ- rity during membrane fusion (Sudhof, 2007). These regulating factors are not capable of handling the many trans-SNARE complexes formed when SNAREs are overexpressed, and vacu- ole lysis results. Lysis of yeast mating pairs. Lysis is also observed during cell fusion of mating yeast. Fusion of haploid cells of opposite mating type yields diploid zygotes (White and Rose, 2001; Chen et al., 2007). The mating reaction begins with phero- mone sensing, which results in cell cycle arrest, polarized growth toward a mating partner (“shmooing”), and induction of a mating-specific transcriptional program. When a polarized shmoo meets a mating partner, their cell walls are woven to- gether and a small channel at the center of the mating pair is cleared, such that the plasma membranes may come into con- tact (Gammie et al., 1998). Membrane fusion rapidly ensues, and further cell wall remodeling and fusion pore expansion al- low for widening of the mating pair neck to allow for nuclear congression and fusion (“karyogamy”). Efficient membrane fusion requires the mating-specific, multipass membrane proteins Prm1p and Fig1p (Heiman and Walter, 2000; Aguilar et al., 2007). Prm1p localizes to the cell surface and is enriched at sites of contact between cells of a mating pair. Its activity is required in only one partner. When PRM1 is deleted in both a and cells, only 40% of mating pairs correctly complete membrane merger and cell fusion. Of the re- maining mating pairs, most arrest at the step of membrane fusion. Cell wall removal continues such that large areas of membrane are in direct apposition, with only 8 nm separating the outer leaflets of the facing plasma membranes. Due to the absence of cell wall at the interface to separate the mating part- ners, the opposed membranes grow and retract such that the cytoplasm of one partner invades the space of the other, forming a membrane-contained structure (“cytoplasmic bubbles”). These cytoplasmic bubble structures are stable; they can grow and re- tract dramatically without losing integrity and allowing mixing between the distinct cytoplasms. The prezygotes remain arrested 182 JCB • VOLUME 183 • NUMBER 2 • 2008 for up to 2 h, by which point individual cells resume the cell cy- cle and begin budding, or repolarize in an attempt to mate with another nearby cell. In addition to fusion failure and extension of cytoplasmic bubbles, a large fraction of prm1 × prm1 mating pairs undergo simultaneous cell lysis (Jin et al., 2004). Like Prm1p, Fig1p is highly enriched at the site of cell fusion (Aguilar et al., 2007). fig1× fig1 mating pairs exhibit many of the same membrane fusion defects as prm1 × prm1 mating pairs, in- cluding cytoplasmic bubbles and simultaneous cell lysis. How- ever, the fig1 defects are milder and less penetrant compared with prm1 mutants and the majority of fig1× fig1 mating pairs are able to complete fusion. The simplest explanation for the lysis and membrane fu- sion defects of prm1 and fgi 1 mutants is that both are caused by misregulation of the cell membrane fusase. The apposed, un- fused cell membranes observed in mating pairs lacking Prm1p and Fig1p provide evidence that the cell fusase is not functioning properly. The concomitant cell lysis defect suggests that the fusase is active but misregulated, generating holes in the cell membranes instead of merging them. Two characteristics of the cell lysis suggest the phenomenon is catalyzed by the cell fusion machinery: the requirement of membrane contact and the timing of the two events. Lysis requires membrane contact, as would activation of the cell–cell fusase. Consistent with this view, dele- tion of FUS1 and FUS2, which results in arrest of mating pairs at the upstream step of cell wall removal, suppresses the prm1 lysis phenotype (Jin et al., 2004). Also, by analyzing many fusion events in a population using time-lapse microscopy, it became evident that lysis events initiate with the same timing as opening of fusion pores in successful mating pairs (Aguilar et al., 2007). Finally, concomitant with mating pair lysis, a small amount of cytoplasmic mixing is observed, consistent with fusion pores opening simultaneously with the appearance of membrane holes that result in mating pair lysis (Aguilar et al., 2007). The extent of prm1 × prm1 mating pair lysis was greatly increased in the absence of extracellular Ca2+, jumping from 20% to 50% of the mating pairs (Aguilar et al., 2007). The in- crease in mating pair lysis in the absence of extracellular Ca2+ is balanced by a similar decrease in mating pair fusion, again sug- gesting the engagement of the fusion machine can have two pos- sible outcomes: productive fusion or lysis. Conversely, prm1 × prm1 mating pair lysis can be suppressed by high concentra- tions of Ca2+. Calcium may play a direct role in the fusion step by interacting with lipid head groups of the opposed bilayers or the proteins that comprise the fusion machinery (Papahadjopoulos et al., 1990). However, wild-type mating pairs do not require calcium to avoid extensive lysis. Alternatively, Ca2+ could pre- vent mating pair lysis by initiating a wound repair process to fix membrane defects initiated by the fusase. In cell culture wound-healing models, membrane holes are repaired by fusion of lysosomal membrane delivery via a Ca2+-dependent mecha- nism that involves the membrane protein synaptotagmin VII (Reddy et al., 2001). Synaptotagmin VII can sense changes in intracellular calcium levels and influence membrane fusion events via calcium and phospholipid binding C2 domains (Rizo and Sudhof, 1998). In yeast, the tricalbin family of proteins has been identified as potential synaptotagmin homologues based