Regulation of Cellular Growth and Division by Ubiquitin-Mediated Proteolysis

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DESCRIPTION (provided by applicant): The cell division cycle is orchestrated by the timed synthesis and subsequent ubiquitin-mediated proteolysis of key regulatory proteins. Two ubiquitin ligase complexes that are known to regulate the cell cycle are the Anaphase Promoting Complex (ARC) and Skp1-Cul1-F-box (SCF) complex. However, there are numerous cell cycle regulators for which the mechanism of turnover remains unknown, and a large number of ubiquitin ligases whose targets have not been identified. To better understand how ubiquitin ligases control the cell cycle, I have developed a method that utilizes a comprehensive library of GFP-fusion proteins and high- throughput microscopy to identify ubiquitin ligase targets in yeast. Previously, I used this technique to identify several targets of one ubiquitin ligase, SCF(Grrl). I will now use this technology to identify the ubiquitin ligases that target the destruction of 75 unstable cell cycle regulators and I will examine the importance of these turnover events for normal cell cycle progression. I identified the transcription factor Tye7 as a target of SCF(Grrl). Tye7 regulates transcription of metabolic genes and is itself transcriptionally regulated throughout the cell cycle. I will determine how Tye7 is targeted for degradation and analyze the consequence of blocking Grr1-mediated turnover. SCF recognizes targets through one of many modular adaptor subunits called F-box proteins. It is unknown how many F-box proteins complex with the SCF at any one time, and whether these complexes change throughout the cell cycle. I will analyze SCF complex composition in different cell cycle and growth states to address these questions. During the mentored phase of the award, I will carry out the proposed screen, which will generate many avenues of research that will be pursued during the independent phase. Moreover, I will learn additional techniques and participate in career development programs that will assist me in becoming and independent scientist and obtaining an independent position. Most cells in the body tightly control growth and division so that they only duplicate when appropriate. When these controls break down and cells divide at incorrect times, this leads to the development of cancer. The cell division cycle is regulated by the timed synthesis and destruction of regulatory molecules. Understanding how timed destruction of these molecules occurs will increase our understanding of processes disrupted in cancer cells.