The fundamental problem addressed in this proposal is the molecular mechanism by which low copy organelles like the yeast vacuole faithfully divide and segregate during cell division. Towards this goal a unique, in vitro assay will be exploited in order to identify novel vacuole membrane proteins functioning in homotypic vacuole fusion. The basis of this technique is in the functional reconstitution of fusion from vacuolar detergent extracts and the fractionation of this extract in order to purify and fully characterize individual proteins which catalyze fusion. Already this technique has successfully been applied to the purification of a fusogenic SNARE complex indicating the feasibility of this approach. Identifying novel membrane components in vacuole fusion bodes well for the more complete understanding of vesicle fusion machinery, of organelle inheritance and its coordination with the cell cycle, and of vesicle trafficking pathways in general. Such important facets of cell biology are clearly related to human health and disease. Hence, yeast vacuole inheritance as a model system may lead to the identification of novel developmental pathways as well as potential therapeutic target sites, each worthy of further scientific investigation.
