The long range goal of the proposed research is to understand the role of ubiquitin conjugation in the regulation of early cell cycle events. This project will have two primary objectives. The first objective will be a biochemical and genetic characterization of the CDC34 ubiquitin-conjugating enzyme of yeast. The second objective will be to identify new proteins that act as regulators or substrates of the CDC34 ubiquitin-conjugating enzyme. The well developed genetics of yeast make it an ideal system in which to determine the importance of biochemically defined protein domains and modification events through mutational analysis. The yeast system is also amenable to the identification of interacting proteins through the isolation of mutations and their subsequent molecular analysis. First, a variety of biochemical techniques will be used to verify the identity of several putative isoforms of the CDC34 protein and to then characterize the structural differences between the isoforms and to use in vitro mutagenesis to determine the importance of any structural differences. Secondly, the genetic interaction known as synthetic lethality will be used as the basis of a screen to isolate mutations in genes encoding proteins that interact as regulators or substrates of the CDC34 enzyme. The mutants will be examined for affects on the cell division cycle and the function of the CDC34 enzyme. A better understanding of ubiquitin metabolism may provide insights into a number of diseases and health-related questions. Ubiquitin is found in the neuronal inclusions associated with several degenerative neurological diseases. It is also found attached to a number of growth factor receptors. Thus an enhanced understanding of ubiquitin function may improve our knowledge in areas ranging from cell growth control to neurodegenerative diseases.
