Figure 1. Models for lipid rearrangements leading to the formation of a fusion pore. The left pathway depicts the classical model for membrane fusion via rupture of a hemifusion diaphragm. Membranes are brought into close apposition (1), the two cis leaﬂets (blue) fuse to form a hemifusion stalk (2), the stalk expands forming a hemi- fusion diaphragm in which trans leaﬂets (green) are in contact (3), and rupture of the hemifusion diaphragm results in a fusion pore (4). In contrast to the classical model for membrane fusion, an alternative pathway, via intermedi- ates drawn on the right, does not always maintain com- partmental identity. Formation of a hemifusion stalk results in the nucleation of holes adjacent to the stalk (3a and 3b), which encircles the holes to form a fusion pore. on their structural similarities (transmembrane anchors coupled to multiple C2 domains) and role in membrane traffic. The C-terminal C2 domains of two tricalbin members, Tcb1p and Tcb3p, exhibit Ca2+-stimulated membrane binding (Schulz and Creutz, 2004). Intriguingly, deletion of TCB3, but not of TCB1 or TCB2, increased prm1 × prm1 mating pair lysis to 50% of mating pairs even in the presence of extracellular Ca2+ (Aguilar et al., 2007). Thus, wound repair processes may mask the true lytic extent of mating in the absence of Prm1p. Viral fusase-induced lysis. Enveloped viruses must fuse with host cells to transfer their genomes. These fusion events are catalyzed by virally encoded transmembrane proteins. A few studies have found that viral fusases create membrane holes concurrent with fusion pore opening. The influenza fusase hemagglutinin (HA) fusion molecule has been studied in many heterologous contexts. During HA- mediated virus–liposome fusion, membrane holes were gener- ated with identical kinetics to lipid mixing, as monitored by the release of large dextran molecules (Shangguan et al., 1996). Similarly, video microscopy revealed content leakage after hem- ifusion diaphragm formation during fusion of HA-expressing fibroblasts with erythrocytes (Blumenthal and Morris, 1999). Finally, conductance measurements during HA-mediated cell– cell fusion showed that membrane permeability increased during early stages of fusion (Frolov et al., 2003). This permability decreased as fusion pores opened, suggesting that membrane leakiness results from membrane rearrangements during pore formation. Thus, it appears that the membrane-destabilizing prop- erties of viral fusases can result in membrane lysis, particularly when they are manipulated to fuse membranes different from the viral envelope and physiologically relevant target cells. In vivo, HIV infection of lymphocytes can result in cell lysis. Cell culture models of this phenomenon showed that cell lysis requires coexpression of the HIV-1 fusase, gp41, and its receptor CD4 (Cao et al., 1996). Curiously, however, the kinetics of lysis initiation are very slow: cells lyse days after maximal gp41 ex- pression is achieved. Using various inhibitors it was demonstrated that the lethal fusase–receptor interaction occurs intracellularly (Madani et al., 2007). In contrast to the fusase-catalyzed cell membrane breaches discussed above, further work is required to elucidate how gp41 activity results in cell lysis. Fusion machines and the pathway of membrane fusion In the prevailing model, membrane fusion does not risk the in- tegrity of compartmental identity (Fig. 1, left pathway). Yet, as described above, leakiness in fusion has been observed in three separate classes of membrane fusion when the balance of fusion players or identity of fusing membranes is altered. These find- ings raise two important questions: Where in the pathway of membrane fusion is lysis initiated, and how is the fusion ma- chinery designed to prevent this outcome? Mechanism of biological membrane fusion. The pathway to membrane fusion must include nonbilayer interme- diates; generating or resolving these intermediates may be the step where the above lysis examples diverge. Recently, a new model for membrane fusion has been proposed in which compartmental identity is temporarily lost (Muller et al., 2003). MEMBRANE LYSIS DURING BIOLOGICAL MEMBRANE FUSION • Engel and Walter 183