| Endocytosis is an essential process in eukaryotic cells in which portions of the plasma membrane and extracellular fluid are internalized, delivered to endosomes, and either recycled back to the plasma membrane or targeted to a degradation compartment. Clathrin-mediated endocytosis (CME) is the best characterized internalization mechanism to date. CME mediates such important processes as the uptake of essential nutrients and the recycling of synaptic vesicles after neurotransmission. Studies have shown that CME is highly dynamic, tightly coordinated, and requires the coupling of multiple subcellular components including lipids and the actin cytoskeleton.; Several proteins—called accessory factors—have been implicated in mediating one or more events during CME. Many of these proteins contain a multi-modular domain organization through which they engage in low affinity interactions with multiple endocytic complex components. These interactions are thought to provide an endocytic protein network that is both stable and dynamic. Though significant advances have provided the atomic structures of several mammalian endocytic proteins, the sequence of intermolecular binding events and how these events are regulated still remains a mystery.; The budding yeast, Saccharomyces cerevisiae, has been used as a model organism for identifying novel endocytic factors that are often highly homologous to endocytic proteins in mammals. My thesis project involved the characterization of two yeast members of a family of clathrin-binding accessory factors called Epsins. Like many endocytic proteins, Epsins are mutli-modular, and the study of their intermolecular interactions has been an important focus in endocytosis research. Early genetic studies implicated the two yeast epsins, Ent1 and Ent2, in an important physiological function, as an ent1Δ ent2Δ double deletion is lethal in yeast. This lethality was mapped to a highly conserved, NH2-terminal segment present in all Epsins. Through genetic and biochemical analysis, we have demonstrated that the yeast Epsins are required for normal endocytosis and actin cytoskeleton organization, bind multiple endocytic machinery components, and are regulated by actin-regulating kinases. Our findings have provided significant insight into the nature and function of Epsins, and more work on these molecules will no doubt be a key factor in understanding endocytic mechanisms in both mammals and yeast. |
