PHOSPHOLIPASE C AND KINETOCHORE FUNCTION

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DESCRIPTION (applicant's description) The long-term goal of our laboratory is to identify and characterize eukaryotic cellular processes regulated by phospholipase C (PLC), an enzyme which plays vital roles in signal transduction pathways. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to produce two important second messengers: inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] which triggers release of calcium from internal stores, and diacylglycerol (DG) which activates the phospholipid- and Ca2+-dependent protein kinase C. Our recent results (Lin et al., 2000) demonstrate that PLC in Saccharomyces cerevisiae (Plc1p protein encoded by the PLC1 gene) associates with kinetochores and affects their ability to bind microtubules. The kinetochore is a specialized organelle that mediates chromosome attachment to spindle microtubules and hence is essential for proper chromosome segregation and cell cycle progression. We found that cells with deletion of PLC1 gene (plc1delta) display higher frequency of chromosome loss, nocodazole sensitivity, and mitotic delay. Furthermore, chromatin extracts from p1c1delta cells exhibit reduced microtubule binding to minichromosomes. However, it remains unknown whether the enzymatic activity of Plc1p is required for proper mitotic function, or whether the mere binding of this protein to kinetochores is sufficient for normal behavior, or whether Plc1p's kinetochore-binding and its enzymatic activity are both required. Our Specific Aims will resolve these alternative possibilities. We will prepare two types of Plc1p mutants: enzymatically inactive mutants with preserved ability to interact with kinetochores, and (ii) mutants which retain enzymatic activity but are unable to interact with kinetochores. Each mutant Plc1p protein will be characterized biochemically and the yeast strains expressing these mutant Plc1p proteins will be fully characterized in terms of fidelity of chromosome transmission, mitotic delay, sensitivity to nocodazole, and the ability of minichromosomes to bind microtubules. The results will lead to better understanding of the molecular mechanisms regulating chromosome segregation during mitosis, cell proliferation, and oncogenesis.