Characterizing the role of adaptor proteins in endocytosis

The plasma membrane (PM) is the barrier between the interior of a cell and the environment, and the composition of this membrane is continuously modified in response to fluctuating conditions. Endocytosis is the process by which portions of the PM and its components are trafficked into the cell, passing through a series of endosomal compartments that ultimately lead to the lysosome in mammalian cells; the lysosome’s yeast organelle equivalent is known as the vacuole. Endocytosis at the PM can occur via multiple mechanisms. In the budding yeast Saccharomyces cerevisiae, the most well understood endocytic pathway is clathrin-mediated endocytosis (CME), and among the earliest players in CME initiation are endocytic adaptor proteins. These adaptors bind to lipids in the PM and proteins, known as cargos, that reside at or within this membrane, as well as other factors involved in CME. In this way, endocytic adaptors link the PM and its contents to the rest of the endocytic machinery. The selection of cargo for internalization is an important part of cellular responses to environmental change, often occurring via interaction of the adaptor with specific amino acid sequences within a cargo. The absence of endocytic adaptors can lead to abnormal phenotypes, such as the accumulation of cargo at the PM. Though the cargo-sorting motifs recognized by some endocytic adaptors have been identified, the mechanism for others, including the muniscin family of adaptors, remains unknown. Additionally, the loss of certain adaptors may place increased importance on the functioning of other proteins in the cell. In this dissertation, we demonstrate that the muniscin adaptor in budding yeast, Syp1, selects cargo for endocytosis via an Asp-x-Tyr motif. We also present results of a visual screen that identified two previously unknown Syp1 cargos, as well as data indicating that Syp1 can direct endocytosis through a clathrin-independent pathway, in addition to CME. We also present findings from a colony-sectoring screen demonstrating that the loss of Vps4, an ATPase necessary for the generation of multivesicular bodies, is lethal in an endocytosis adaptor mutant strain. Finally, we provide evidence that upregulation of endocytic recycling is responsible for this negative interaction