Checkpoints regulate the cell cycle by delaying progression until the faithful completion of a prior step. A morphogenesis checkpoint has recently been revealed in Saccharomyces cerevisiae that delays the onset of mitosis until the formation of a bud, thus avoiding the generation of cells with multiple nuclei. The delay in mitosis is a result of the tyr19 phosphorylation of the master regulatory cyclin dependent kinase, Cdc28p. The goal of this research is to begin to dissect the molecular nature of the morphogenesis checkpoint, primarily by identifying proteins functioning in this checkpoint. This goal will be approached in two independent ways. The first approach will be to start at Cdc28p and work back through the checkpoint. The activity of Swe1p, a kinase which phosphorylates Cdc28p tyr19 will be assayed in cells in which budding has been impaired. Preliminary results suggest that posttranslational modification of Swe1p affects Swe1p kinase activity and may play a major role in the checkpoint. If this is the case, proteins will then be identified which affect the activity of Swe1p. These proteins will then be assayed to determine if they are also regulated by the checkpoint. If so, how are these proteins regulated and by what? Using this approach, proteins involved in this checkpoint will be identified step by step, beginning at Cdc28p. The second approach will be to identify proteins involved in the checkpoint by a genetic screen. Swe1p is an essential protein for the operation of this checkpoint, hence swe1 is a morphogenesis checkpoint mutant. Based on the phenotypes of swe1 in combination with a mutant defective in the formation of a bud, a genetic screen has been devised to uncover other morphogenesis checkpoint mutants. These checkpoint mutants will be cloned and further analyzed to determine what role they play in the morphogenesis checkpoint.
