The Identification and Characterization of Proteins Required for Endocytosis in The Budding Yeast Saccharomyces Cerevisiae
All eukaryotic cells are surrounded by a plasma membrane that segregates the interior of the cell from the extracellular environment. Transport to and from this membrane regulates homeostasis of the membrane itself, allows the import of material from the extracellular space, and provides a mechanism for communication with the outer environment. The process of endocytosis describes the internalization and subsequent transport of plasma membrane components and extracellular material. Endocytosis requires the coordinated activity of many protein factors, and the specific mechanisms employed by the cell for internalization remain only partially understood. The studies presented in this work focus on identifying novel proteins involved in endocytosis and characterizing their role in membrane trafficking. An in vitro endocytosis assay was adapted for use with yeast cytosol, in order to provide a biochemical method for identifying proteins required for the internalization of coated pits or endocytic vesicles. However, the combination of yeast cytosol with mammalian membranes failed to induce physiologically significant internalization. Through a genetic screen for mutants defective for the uptake of a lipophilic dye, an allele of the FAB] lipid kinase was identified. The characterization of this allele through the analysis of internalization of a variety of cargo revealed a role for PtdIns(3,5)P2 in sorting and trafficking of internalized proteins at the late endosome. Finally, a spontaneous reversion screen of a temperature sensitive allele of the yeast epsin ENT1 revealed residues within the ENTH domain that are required for maintaining viability in yeast cells. These residues are also important for an interaction between the ENTH domain and GTPase activating proteins (GAPs) for the master regulator of polarity Cdc42. Phenotypic analysis of this temperature sensitive allele revealed that the disruption of the ENTH GAP interaction leads to defects in polarity-dependent processes such as budding, mating projection formation, cell wall integrity, actin cytoskeleton polarity, and actin cable formation. Together, these studies have revealed previously unknown roles for proteins in endocytosis that demonstrate the myriad interconnections within membrane trafficking pathways and between processes, such as endocytosis and cell polarity, that are necessary for cell physiology.