Regulation of ART-mediated plasma membrane protein degradation in yeast

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Plasma membrane proteins control interactions between the cell and its environment, governing virtually every aspect of cellular biology. Cells regulate both the delivery of proteins to the plasma membrane and the removal of proteins from the plasma membrane in order to control the abundance and activity of proteins at the cell surface. The primary mechanism for plasma membrane protein removal is their endocytic uptake, which is initiated and controlled by cargo-ubiquitination. Proper regulation of the protein composition at the cell surface is critical to cell survival and human health and disease. But how does a cell manage to integrate intracellular and extracellular signals and choose one specific plasma membrane protein among the hundreds of others for degradation? One intriguing explanation is the existence of a family of arrestin-like adaptor proteins, called ARTs in yeast, which recruit the respective ubiquitin ligase specifically to proteins destined for degradation. Recent studies suggest that endocytic protein degradation is mainly regulated on the adaptor protein level. However, the molecular mechanisms governing ART activity are only poorly understood and, thus, will be addressed in this study. Using a genome-wide overexpression approach in the baker's yeast Saccharomyces cerevisiae, I will screen for signaling pathways that influence ART activity. This will extend our understanding of how cells integrate several signaling pathways into coordinated protein degradation. It is already known that ART activity is modulated by phosphorylation. Recent mass-spectrometry data shows that ARTs are extensively phosphorylated. However, only few phosphorylation sites are characterized in respect to their regulatory function. Thus, I will mutagenize poorly characterized phosphorylation sites and characterize the mutants biochemically and cell-biologically in respect to their activity. In particular, I want to elucidate whether ART phosphorylation influences their ability to bind target proteins. This study will improve our understanding of the regulation of endocytic protein degradation.