Simulations of membrane fusion using coarse-grained lattice models predicted that the stalk intermediate promotes the forma- tion of adjacent holes in the bilayers (Fig. 1, stage 3a and 3b). These holes are then surrounded by the stalk to form a fusion pore (3b). This pathway is less energetically costly than the tra- ditional, nonleaky hemifusion hypothesis (Katsov et al., 2006). Lysis could emerge from this pathway if these membrane holes expand before the stalk can encircle them to form the fusion pore. A similar leaky structure would be created if, instead of bi- layer rupture within the hemifusion diaphragm, a hole opens in one of the two bilayers adjacent to the hemifusion diaphragm. Alternatively, lysis may occur before formation of the hemifusion stalk as a consequence of trying to transition to the nonbilayer intermediate. Strongly bending membranes may be a strategy for destabilizing bilayers such that they will form a stalk intermediate (Kozlov and Chernomordik, 1998). This could be a risky endeavor—generation of unstable, highly curved membranes could result in membrane rupture. Assembling a fusion machine. Viral fusases and SNAREs are sufficient to fuse lipid bilayers and biological membranes, yet this feat is not achieved by a single HA trimer or trans-SNARE pair. Instead, these proteins are assembled into a greater fusion machine, consisting of multiple core fusases (i.e., HA, gp41, a trans-SNARE pair) and, in most cases, regulatory proteins (i.e., HOPS, synaptotagmin, complexin) (Tang et al., 2006). Additionally, lipids act as regulators and facilitators of membrane fusion, recruiting fusase subunits and allowing highly curved membrane intermediates (Fratti et al., 2004; Chernomordik and Kozlov, 2008). Core fusase regulators have been described to govern specificity and timing of the fusion event. Might others ensure membrane integrity during lipid re- arrangement? The characteristics of this fusion machine realize the fusogenic, and may limit the lytic potential of core fusases. Both viral and intracellular fusion use the concerted action of multiple fusases to achieve the energy required for membrane fusion. Kinetic analysis of fusion by cells expressing HA with different surface densities estimated a minimum of three HA tri- mers mediate membrane fusion (Danieli et al., 1996), and mod- eling has suggested that the concerted action of at least eight HA trimers, including two in the activated state, are required to open a fusion pore (Bentz, 2000). In addition to recruiting multiple fusases, the geometry of their association is likely important for efficacy of the fusion machine. The geometry of the HA fusion machine is thought to be circular and to surround the hemifusion stalk and nascent fusion pore (Chernomordik et al., 1998). Multiple trans-SNARE pairs are required to achieve fusion, and atomic force microscopy showed that SNAREs also associate in a ring-like fashion (Hua and Scheller, 2001; Cho et al., 2002; Hofmann et al., 2006). If a fusion machine were haphazardly assembled, the membrane-destabilizing activities of the core fusases may result in membrane lysis instead of a fusion pore. The geometrical information behind HA oligomerization is likely inherent in the molecule, but this may not be the case for SNAREs or the as-yet unidentified yeast fusase, allowing for the possibility of fusase organization by an independent protein fac- tor. At a gross localization level, both the Rab Ypt7p and regula- tory lipids are required for the enrichement of SNAREs at the 184 JCB • VOLUME 183 • NUMBER 2 • 2008 Figure 2. Models for regulation of fusion integrity by nonfusase factors. Fusase molecules are drawn in green, integrity promoting factors in red. (A) Regulation of lytic potential by organizing fusase molecules. Architec- tural factors recruit core fusases into a ring-shaped fusion machine, which guides the membrane-destabilizing activity of the core fusases toward fusion pore formation (top). In the absence of these factors, core fusase activity is not geometrically coordinated, resulting in membrane rupture (bottom). (B) Restriction of membrane hole expansion by a ring of mem- brane proteins. If the pathway to membrane fusion were inherently leaky (see Fig. 1), the risks of membrane hole expansion may be mitigated by protein factors surrounding the nascent fusion pore (top). In their ab- sence, hole expansion may proceed and result in loss of compartmental integrity (bottom). vertex ring of contacting vacuoles (Wang et al., 2003; Fratti et al., 2004). Furthermore, the Ypt7p effector HOPS directly in- teracts with the SNARE Vam7p, stimulates trans-SNARE com- plex formation, and proofreads trans-SNARE pairs (Stroupe et al., 2006; Collins and Wickner, 2007; Starai et al., 2008). Despite the intimate relationship between HOPS and vacuolar SNAREs, HOPS does not limit vacuole lysis driven by high