DETERMINATION OF 4 BIOCHEMICALLY DISTINCT, SEQUENTIAL STAGES DURING VACUOLE INHERITANCE IN-VITRO

Vacuole inheritance in Saccharomyces cerevisiae can be reconstituted i n vitro using isolated organelles, cytosol, and ATP. Using the require ments of the reaction and its susceptibility to inhibitors, we have di vided the in vitro reaction into four biochemically distinct, sequenti al subreactions. Stage I requires exposure of vacuoles to solutions of moderate ionic strength. Stage II requires ''stage I'' vacuoles and c ytosol. In stage m, stage II vacuoles react with ATP. Finally, during stage IV, stage III vacuoles at a certain, minimal concentration compl ete the fusion reaction without further requirement for any soluble co mponents. Reagents that inhibit the overall vacuole inheritance reacti on block distinct stages. Stage III of the reaction is sensitive to th e proton ionophore CCCP, to inhibitors of the vacuolar ATPase such as bafilomycin A(1), and to the ATP-hydrolyzing enzyme apyrase, suggestin g that an electrochemical potential across the vacuolar membrane is re quired during this stage. Inhibition studies with the amphiphilic pept ide mastoparan and GTP gamma S suggest that GTP-hydrolyzing proteins m ight also be involved during this stage. Microcystin-LR, a specific in hibitor 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 ma y allow the development of specific assays for the factors which catal yze each of the consecutive steps of the in vitro reaction.