9727240 McDonald Accurate cell division is fundamental to the growth and development of all organisms. For genetic information to be faithfully transmitted to a cell's progeny, its chromosomes must be correctly duplicated and then precisely segregated to each of the two new daughter cells. The broad aim of the work to be done in this project is to gain a better understanding of the formation of the mitotic spindle, which is the intracellular structure that functions to segregate the duplicated sets of chromosomes. The spindle apparatus is organized at each of its two ends by a structure, termed the centrosome (in animal cells) or spindle pole body (in yeast cells). Since the spindle requires two centrosomes for proper organization, the first step of spindle formation is duplication of the centrosome. The work to be done in this project will address the mechanism of centrosome duplication. In the yeast, Saccharomyces cerevisiae, several genes have been shown to be specifically required for spindle pole body (SPB) duplication. One of these genes, PCS1, encodes a protein (Pcs1p)that is part of the proteasome, a multiprotein complex which functions to proteolytically degrade certain proteins that are destined for such degradation. Specifically, Pcs1p is a subunit of the proteasome cap, which is believed to have a gatekeeping function of controlling the entry of proteins that will be degraded. This intriguing observation suggests that Pcs1p specifically recognizes some substrate(s) protein(s) whose degradation is required in order for SPB duplication to occur. The experiments to be performed in this project will test this hypothesis by searching for the proteins whose degradation triggers SPB duplication. Three screens, combining the techniques of genetics, molecular biology, and biochemistry, will be undertaken to search for these proteins. Extragenic loss-of-function suppressors of the pcs1 mutant phenotype will be sought, on the theory that loss (by mutation) of such substrate proteins might compensate for lack of Pcs1p-mediated degradation. Based on similar logic, a screen for genes that enhance the pcs1 phenotype when overexpressed will also be performed. Third, a phage-display library of yeast protein fragments will be screened for Pcs1p-binding constituents, or if this is not productive, a two-hybrid screen for Pcs1p-interacting proteins will be undertaken. Candidate proteins identified in these screens will be further characterized to determine if they do indeed undergo Pcs1p-mediated proteolysis and if so, if this proteolysis is cell cycle stage-specific. Given the high level of similarity between the intracellular processes in yeast, plants and animals, these studies have great potential to clarify the process of centrosome duplication in all eukaryotic organisms.
