DESCRIPTION (provided by applicant): Due to its involvement in the control of a wide array of cellular processes, substrate-selective protein degradation has emerged as a major cell regulatory mechanism. Permanent or transient metabolic instability is a property of a number of different regulatory proteins, including transcription factors, signaling molecules, cell cycle regulators, apoptotic factors, proto-oncogene products, and tumor suppressor proteins. In eukaryotes, these regulatory proteins are targeted for destruction by a complex enzymatic system that involves their post-translational modification by ubiquitin and their subsequent degradation by a large multi- subunit protease called the proteasome. Previously, we determined that the rapid ubiquitin-dependent degradation of a master regulatory transcription factor, which is a key determinant of cellular identity in the yeast Saccharomyces cerevisiae, is required for yeast cells to change their phenotype during a normal differentiation event. To further our understanding of how a cell exploits the process of ubiquitin-mediated proteolysis to change patterns of gene expression and switch between alternative phenotypic states, we propose to characterize the biological impact and the molecular mechanisms of the rapid proteolysis of two different yeast cell type-specific transcription factors, alpha1 and alpha2. Our overall objective is to understand the mechanisms by which the rapid degradation of these multiple master regulatory transcription factors is synthesized into a single phenotypically defined developmental transition in yeast. Specifically, we will (1) characterize the interplay between the proteolysis of alpha2, the dynamics of alpha2 binding to target gene promoters and alpha2-mediated repression in vivo; (2) identify the molecular features of the cell type regulator alpha1 that govern its stability; and (3) determine the enzymatic pathways and cellular machinery that cause alpha1 to be short-lived. These studies, which characterize phenotypic switching events in an experimentally amenable model system, will provide information for understanding not only the differentiation processes that underlie normal cell growth and development, but also the pathological situations caused by defects in switching from a state of proliferation to the fully differentiated, non-proliferating cell-type.