VACUOLE DIVISION/SEGREGATION DURING YEAST CELL CYCLE

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The division and segregation of cytoplasmic organelles must be spatially and cell-cycle regulated to insure faithful inheritance through many cell divisions. Study of this process for the vacuole (lysosome) of S. cerevisiae during the last 5 years has 1. established the cytology of its division and inheritance pattern 2. led to vac mutants which are specifically defective in vacuole inheritance, and 3. provided an in vitro reaction, the formation of vacuole-derived tubulo-vesicular protuberances, which faithfully reflects the in vivo inheritance process. This in vitro reaction requires semi-intact cells or isolated vacuoles, cytosol, ATP, GTP, and incubation at 23 to 37 degrees C and depends on tubulin and the VAC1 and VAC2 proteins. Planned studies will combine this cytology, genetics and biochemistry. New, and old, vac mutants will be collected, combined genetically to explore interrelationships, VAC genes cloned and sequenced, and antibody prepared for immunolocalization and to aid in biochemical assay. The components of the in vitro reaction will be fractionated and purified, using complementation of vac extracts, nucleotide affinity matrices, and total fractionation of the yeast cytosol, assaying for the fractions required to support tubulovesicle formation. The relationships of this reaction to proteins such as cyclins and kinases which govern the cell cycle and to MAPS such as kinesin and dynein which drive microtubule-based organelle motion will be explored. These studies will provide basic information on an important, yet little studied, aspect of cell division.