Before this work, Prm1 was the only protein known to act at the plasma membrane fusion step during yeast mating. Here, we expand the cast of players in membrane fusion with the characterization of . FIG 1
was initially identified as a pheromone-induced gene with roles in cell polarization and fusion (Erdman et al., 1998
). Here, we show that Fig1 localizes to the zone of cell–cell contact and that deletion of FIG 1
results in a membrane fusion defect after cell wall removal, as indicated by ultrastructural analyses and the formation of cytoplasmic bubbles that are bounded by tightly apposed plasma membranes from both mating partners. Moreover, bilateral deletion of FIG 1
significantly enhances the fusion defects observed in prm1
Δ × prm1
Δ mating pairs. Unlike wild-type and prm1
Δ × prm1
Δ mating pairs
Δ × fig1
Δ mating pairs are delayed in initiating of fusion but the delay is shorter than that observed in fus1
Δ × fus1
Δ mating pairs and, unlike fus1
Δ × fus1
Δ mating pairs, fig1
Δ × fig1
Δ mating pairs still cluster vesicles at the zone of cell fusion (Aguilar, unpublished data). Although fig1
Δ × fig1
Δ mating reactions are sensitive to the removal of Ca2+
, Fig1 most likely has fusion promoting roles independent of Ca2+
influx because removal of extracellular calcium from wild-type mating pairs does not result in mating defects. As a member of the Claudin superfamily, Fig1 may share functional properties with tight junction proteins and possibly help to arrange the fusion machinery by holding membranes in proximity (Van Itallie and Anderson, 2004
; Zhang et al., 2006
Albeit severely compromised, some residual fusion activity remains in the absence of Prm1 and . This observation suggests that either 1) Prm1 and Fig1 are important yet nonessential components of the fusion machinery or that 2) an alternate Prm1- and Fig1-independent fusion pathway(s) can compensate for their absence. Currently available data do not allow us to distinguish between these possibilities.
Nonproductive mating pairs that fail to fuse in the absence of Prm1 and/or Fig1 can either lyse or remain unfused with their plasma membranes in close apposition. It was previously suggested that the observed cell lysis may be a direct result of engagement of the cell fusion machinery and possibly be intrinsically linked to the mechanism of lipid bilayer fusion (Jin et al., 2004
); the results presented here support this view. In , we describe the relationship between the two phenotypes of mating pairs lacking Prm1 and/or Fig1 by quantifying to values, the “activity” and “fidelity” of the membrane fusion machinery. We define activity as the probability of engagement of an active membrane fusase, which can lead to either fusion or lysis. Only 62% of prm1
Δ × prm1
Δ mating pairs engage a fusase, compared with 99% for wild-type mating pairs (). We define fidelity as the probability that cells in a mating pair will survive after engagement of a fusase. Fidelity declines from 96% in wild-type mating pairs to 63 and 43% in mating pairs missing Prm1, and Prm1 and Fig1 in both mating partners, respectively. Thus, only 37% of cells missing Prm1 and Fig1 engage the fusion machinery and of those that do only 43% survive.
Influence of Ca2+ on activity and fidelity values
In contrast to fusase activity, fusase fidelity is sensitive to the state of extracellular Ca2+. Whereas we found no requirement for Ca2+ during mating of wild-type cells, fidelity values for mating reactions carried out in the absence of Ca2+ drop to 7 and 1.2% for prm1Δ × prm1Δ and prm1Δ fig1Δ × prm1Δ fig1Δ mating pairs, respectively. Thus, Ca2+ masks the true extent of the fidelity defects of prm1 and fig1 mutant fusion machines, but it is important only in the context of the defective fusion machine in the mutant cells.
Our results support in multiple ways a functional coupling of lysis to the engagement of the fusion machine: First, by removing Ca2+
to favor lysis, we observe that the timing of lysis events is the same as the timing of fusion. Second, we demonstrate that the two cells of a mating pair lyse synchronously, as expected for events at the interface between both cells in a mating pair. Third, mixing of cytoplasmic contents occurs concomitant with the initiation of lysis. This implies that lysis is initiated as fusion is catalyzed, most simply explained by hypothesizing a common machinery for the two outcomes. It is possible, for example, that a defective fusion machinery may not contain the fusion zone properly or correctly resolve unstable membrane intermediates, leading to mating pair lysis. Indeed, recent models of bilayer fusion (Muller et al., 2003b
) pose that membrane fusionlysis is not a failsafe process: formation of the lipid stalk favors the formation of holes adjacent to the stalk in each of the two engaged membranes. Thus, it is conceivable that the very act of bilayer membrane fusion can cause membrane rupture and cell lysis—unless the fusion zone is contained by accessory proteins of the fusion machinery. Prm1 and Fig1 could play such a role, for example, by providing a molecular fence that corrals the fusion zone and prevents the catastrophic spread of local membrane damage. Corralling also could serve an instructive role helping organizing the activity of the fusion machine, thus explaining the reduced fusion activity in mating reactions of cells lacking Prm1 and Fig1.
In this light, an attractive explanation for the Ca2+ effect in the mutant cells is that the mutations enhance lysis, which is counteracted by Ca2+-dependent membrane repair mechanisms, thus influencing the fusion–lysis balance by rescuing potential lysis events. This would explain why Ca2+ is not required during wild-type mating reactions where Prm1 and Fig1 prevent lysis events from occurring. We provide evidence that Tcb3, a yeast synaptotagmin orthologue, may function as a Ca2+ sensor in a membrane repair pathway operating during this process. Deletion of TCB3 mimics the lysis increase observed in prm1Δ × prm1Δ mating pairs upon Ca2+ depletion. This model also would explain the accumulation of membranes in prm1Δ × prm1Δ mating pairs upon Ca2+ depletion.
Although attractive, this model leaves many interesting questions to be solved: For example it does not explain all of the observed effects that Ca2+
exerts on membrane fusion. In particular, the observation that high Ca2+
concentrations partially suppress the defects of prm1
Δ × prm1
Δ mating pairs suggests that Ca2+
at high concentrations also may promote the fusion of apposed membranes, perhaps by directly interacting with membrane lipids as seen in membrane fusions assays of pure lipid vesicles (Duzgunes et al., 1981
; Ellens et al., 1985
). Alternatively, other yet to be identified Ca2+
sensors in addition to Tcb3 may participate in the fusion process.