Understanding the mechanism of membrane fusion: How does the vacuolar tethering complex HOPS initiate the first contact between membranes? And by which means does it integrate SNARE, Rab, and lipidic functions in membrane fusion?

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Eukaryotic cells are compartmentalized by their endomembrane system. This spatial separation of cellular functions prevents undesired interference between different metabolic pathways, and necessitates the regulated transport of proteins, lipids, and solutes between organelles. Small vesicles containing cargo molecules bud from each donor compartment, travel across the cytosol, and specifically dock and fuse with the membrane of the target organelle. Yeast vacuole fusion is a perfectly established model to study this process of membrane fusion. As the core components of this fusion machinery have been discovered, it is now possible to address a series of questions on how they mechanistically achieve the merging of two distinct membranes. This proposal focuses on the initial step of membrane fusion: "tethering". The analysis of other membrane fusion systems revealed a great structural and functional variability of the tethering complexes. For yeast vacuole fusion, I will determine how the tethering complex HOPS initiates the first contacts between membranes prior to fusion. To this end, I intend to:(1) Determine the stoichiometric composition of the fundamental tethering unit.(2) Employ cross-linking techniques to map the direct molecular interactions that underlie tethering.(3) Establish a novel tethering assay to quantify the dynamics of tethering and the role(s) of other factors such as phosphoinositides and SNAREs in this process.(4) Quantify the kinetic constants of the tethering interactions to determine whether there is synergy amongst the individual components.(5) Relate these interactions to the overall fusion process through both in-vitro and in-vivo assays.