In the budding yeast Saccharomyces cerevisiae, vacuoles are inherited by the projection of vesicles and tubules from the mother‐cell vacuole into the growing daughter cell during the S phase. These vesicles then fuse and form the daughter‐cell organelle. We have described previously in vitro reactions of the formation of vacuole‐derived segregation structures and of vacuole‐vacuole fusion. Homotypic vacuole fusion requires cytosol, ATP and a physiological temperature, and is sensitive to GTPase inhibitors. These reactions are divisible into early stages which require ATP and cytosol, and late stages which require neither. Here, we report that Ypt7p, a ras‐like GTPase implicated previously in endocytosis in yeast, is largely localized to the vacuole and is required on both partners during the in vitro vacuole fusion reaction. The in vitro fusion reaction is inhibited either by Gdi1p, which extracts the GDP‐bound form of ras‐like GTPases from membranes, or by antibodies specific for Ypt7p. The presence of anti‐Ypt7p during the early stages of the reaction inhibits the development of cytosol‐ and ATP‐independent intermediates. Although cytosol and ATP are no longer needed for the late stage of vacuole inheritance in vitro, the inhibition of this late stage by anti‐Ypt7p or Gdi1p requires the continued presence of ATP and cytosol. Ypt7p is the first GTPase for which a direct role in organelle inheritance has been established.

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Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) are important for fusion. Monoclonal antibodies to PI(4,5)P 2 , neomycin (a phosphoinositide ligand), and phosphatidylinositol-specific phospholipase C interfere with the reaction. Readdition of PI(4,5)P 2 restores fusion in each case. Phosphatidylinositol 3-phosphate and PI(3,5)P 2 synthesis are not required. PI(4,5)P 2 is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec17p (␣-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement identified for vacuole fusion so far. Furthermore, PI(4,5)P 2 is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P 2 controls two steps of vacuole fusion. INTRODUCTIONYeast vacuoles are very dynamic organelles. Their number, size, and shape change not only when vacuoles are transmitted to growing daughter cells, but also when the source of nutrient or other environmental factors (e.g., osmotic conditions) change (Wiemken et al., 1970; Wickner, 1988, Conradt et al., 1992;Bone et al., 1998). The changes in number and size can readily be explained by cycles of fragmentation and fusion of vacuoles. A cell free system for the fusion of vacuoles from Saccharomyces cerevisiae has been developed, allowing both morphological and biochemical assays of the reaction (Conradt et al., 1992). Fusion can be quantified conveniently via the proteolytic cleavage and activation of proalkaline phosphatase (located at the luminal side of the vacuolar membrane) by the vacuolar proteinase A . Using this assay, vacuole fusion could be kinetically dissected into several steps: priming, tethering, docking, and fusion Mayer et al., 1996;Mayer and Wickner, 1997;Ungermann et al., 1998). Priming, tethering, and docking require the ATPdependent activation of SNAREs through Sec17p (␣-SNAP) and Sec18p (NSF) as well as the rab-like GTPase Ypt7p and LMA1 (Xu and Wickner, 1995;Mayer and Wickner, 1997;Xu et al., 1997Xu et al., , 1998Ungermann et al., 1998). They lead to the formation of v/t-SNARE complexes in trans, i.e., between separate vacuoles, and to the stable association of the membranes.Phosphatidylinositol phosphates have become a focus of research on intracellular trafficking (for review, see De Camilli et al., 1996;Shepherd et al., 1996;Martin, 1998). These phospholipids have been implicated in multiple trafficking steps and were shown to associate with and regulate different components required for these steps: e.g., the regulation of the activity of small GTPases and their cofacto...

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