Vacuole inheritance in Saccharomyces cerevisiae can be reconstituted in vitro using isolated organelles, cytosol, and ATP. Using the requirements of the reaction and its susceptibility to inhibitors, we have divided the in vitro reaction into four biochemically distinct, sequential subreactions. Stage I requires exposure of vacuoles to solutions of moderate ionic strength. Stage II requires "stage I" vacuoles and cytosol. In stage III, stage II vacuoles react with ATP. Finally, during stage IV, stage III vacuoles at a certain, minimal concentration complete the fusion reaction without further requirement for any soluble components. Reagents that inhibit the overall vacuole inheritance reaction block distinct stages. Stage III of the reaction is sensitive to the proton ionophore CCCP, to inhibitors of the vacuolar ATPase such as bafilomycin A1, and to the ATP-hydrolyzing enzyme apyrase, suggesting that an electrochemical potential across the vacuolar membrane is required during this stage. Inhibition studies with the amphiphilic peptide mastoparan and GTP gamma S suggest that GTP-hydrolyzing proteins might also be involved during this stage. Microcystin-LR, a specific inhibitor of protein phosphatases of type 1 and 2A, inhibits stage IV of the inheritance reaction, indicating that a protein dephosphorylation event is necessary for fusion. The definition of these four stages may allow the development of specific assays for the factors which catalyze each of the consecutive steps of the in vitro reaction.

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