The coordinated transition between the stages of the eukaryotic cell cycle is controlled by a number of regulatory mechanisms at both the transcriptional and post-translational levels Errors in the regulation of the cell cycle lead to genetic instability and play a critical role in the onset and progression of many cancers This proposal investigates the post-translational regulation of cell cycle related proteins in S cerevisiae by utilizing a proteomics approach A fusion library will be created in which all known ORFs me tagged with a common epitope while keeping their expression under the control of their natural promoters The library will allow for endogenous protein levels to be quantified through the immunodetection of the fused tag. High throughput protocols will be developed in order to detect changes in the levels of all expressed proteins, as a function of the cell cycle, in 96-well format. This analysis will be used to identify, classify and study new cell cycle related proteins and to discover novel post-translational regulatory mechanisms. More detailed follow-up studies will provide further clues as to the exact role of the identified proteins. Also, the correlation between changes in mRNA and protein levels during the cell cycle will be investigated. The global-scale analysis of post-translational regulatory mechanisms will add to our understanding of cell cycle regulation and allow for the identification of novel genes that have relevance to the study of cancer More generally, it is hoped that the fusion library-based proteomic approach, employed here in the study the cell cycle, will in future serve as a general model for the investigation of other complex biological systems
