Regulation of Cellular Growth and Division by Ubiquitin-Mediated Proteolysis

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

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 (APC) 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, we developed a method that utilizes a library of strains expressing GFP-fusion proteins and highthroughput microscopy to identify ubiquitin ligase targets in yeast, and are using this approach to identify the ubiquitin ligases that target 73 unstable cell cycle regulators for destruction. In the experiments proposed here, we will determine the mechanism of turnover of these cell cycle proteins by specific E3s, and examine the importance of these turnover events for nonnal cell cycle progression. The SCF(Gn'l) ubiquitin ligase is known to target both cell cycle and metabolic regulators for destruction. We previously identified the transcription factor Tye7 as a SCF(Grrl) target. Tye7 regulates transcription of metabolic genes and is itself transcriptionally regulated throughout the cell cycle. We will detennine Tye7 is targeted for degradation and analyze the consequence of blocking SCF-mediated turnover. Finally, we will investigate how the cell cycle and metabolic factors influence SCF ligase activity. SCF ligases recognize 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. We will analyze SCF complex composition in different cell cycle and growth states to address these questions.