Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was required for efficient lumenal compartment mixing. While the physiological HOPS tethering complex caused a few-fold increase of trans-SNARE association, the rate of content mixing increased more than 100-fold. Thus tethering has a role in promoting membrane fusion that extends beyond simply increasing the amount of total trans-SNARE complex.
Cells of higher organisms contain compartments called organelles and structures called vesicles that transfer molecules and proteins between these organelles. Each organelle and each vesicle is enclosed within a membrane, and these membranes must fuse together to allow these transfers to take place. A certain group of proteins, called SNAREs, have a central role in these fusion events.
Since membrane fusion is difficult to observe directly, many researchers have used a method called ‘fluorescent lipid dequenching’ to study it indirectly. In this approach, one fraction of vesicles is labeled with two fluorescent molecules, with one of these molecules quenching the fluorescence of the other. However, when a labeled vesicle fuses with an unlabeled vesicle, the surface concentrations of the fluorescent molecules are diluted. This reduces the amount of quenching and the resulting increase in fluorescence can be measured.
Experiments utilizing this technique had suggested that SNARE proteins are sufficient for fusion to take place, and that no other protein complexes need to be present. However, when a different assay method called ‘lumenal compartment mixing’ was used, little fusion was seen when the only proteins present were the SNAREs. The lumenal compartment mixing approach relies on measuring the degree of mixing between the contents of two vesicles.
To address these conflicting results, Zick and Wickner used both methods to study fusion in a yeast-based system. The lumenal compartment mixing approach, which is the more reliable method, revealed that rapid and efficient membrane fusion in fact requires another protein complex, called HOPS, to hold the two membrane vesicles together.
Zick and Wickner found that the HOPS complex does not enable fusion by just increasing the amount of interactions between the SNARE proteins. Rather, it seems to facilitate the formation of a particular quality of SNARE interactions. Future work is needed to work out how the SNARE complexes become ‘fusion-competent’, and to explore the mechanism that allows the HOPS complex to assist in the formation of fusion-competent SNARE complexes.