When cells divide, cytoplasmic organelles are donated from the mother cell to the daughter cells. Little is known about how organelle size and number are determined, how organelle growth and division are coordinated with the cell-cycle, or how organelles move and arrive at their correct locations in the cell. To address these questions, vacuole partitioning in the budding yeast Saccharomyces cerevisiae is being studied. During growth, a portion of the mother cell vacuole, the segregation structure, migrates into the bud. This process, in which the vacuole travels along a specific track, is rapid (0.1-0.2 mu/s) and is coordinated with the cell cycle. From a new screen for yeast mutants defective in vacuole segregation (vac), 14 complementation groups have been identified. These mutants may be divided into three classes, each representing a distinct step in the pathway. Class I vac mutants reveal the role of actin-based motility in vacuole inheritance. Among the Class I mutants are vac15/myo2-2(mutated in MYO2, a class V myosin) and a subset of specific act1 (actin) alleles. Another Class I mutant, vac8, led to the discovery of a vacuole membrane protein with armadillo repeats, Vac8p, that may link the membrane to actin. Interactions between the vacuole membrane and actin will be studied. Class III Vac proteins are required for a late step in vacuole segregation and are defective in vacuole membrane scission. Among these are Vac7p and Vac14p, which show no significant homology with known proteins. However, based on the phenotypic similarities of vac7, vac14, and fab1 mutants, the functions of these proteins may involve the newly- identified phosphatidylinositol 3,5-bis phosphate. Thus, studies of the Class III genes and their protein products will address the role of phosphatidylinositol 3,5-bis phosphate in vacuole membrane scission. The proposed research seeks to 1) elucidate the functions of Vac8p; 2) study the other Class I mutants whose phenotypes suggest that they are defective in vacuole membrane-actin interactions; 3) develop in vitro assays for vacuole membrane-actin association and movement; 4) explore the relationship between phosphatidylinositol 3,5-bis phosphate metabolism and vacuole membrane scission.