Regulation of ART-mediated plasma membrane protein degradation in yeast

质膜蛋白控制着细胞与其环境之间的相互作用，几乎控制着细胞生物学的每一个方面。细胞通过调节蛋白质向质膜的传递和从质膜上去除蛋白质来控制细胞表面蛋白质的丰度和活性。质膜蛋白去除的主要机制是它们的内吞摄取，这是由泛素化启动和控制的。适当调节细胞表面的蛋白质组成对细胞存活和人类健康和疾病至关重要。但是，细胞是如何整合细胞内和细胞外信号，并在数百种质膜蛋白中选择一种特定的蛋白进行降解的呢？一种有趣的解释是，存在一种类似抑制素的衔接蛋白家族，在酵母中被称为ARTs，它将各自的泛素连接酶专门招募到注定要降解的蛋白质上。近年来的研究表明，内吞蛋白的降解主要受受体蛋白水平的调控。然而，控制ART活性的分子机制尚不清楚，因此将在本研究中加以解决。使用面包师酵母的全基因组过表达方法，我将筛选影响ART活性的信号通路。这将扩展我们对细胞如何整合几种信号通路以协调蛋白质降解的理解。我们已经知道抗逆转录病毒活性是由磷酸化调节的。最近的质谱数据显示，ARTs被广泛磷酸化。然而，只有少数磷酸化位点在其调节功能方面具有特征。因此，我将诱变特征不佳的磷酸化位点，并在生物化学和细胞生物学方面对突变体的活性进行表征。特别是，我想阐明ART磷酸化是否影响它们结合靶蛋白的能力。本研究将提高我们对内吞蛋白降解调控的认识。

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.