Defining the Essential and Endocytic Functions of Pan1 in Saccharomyces cerevisiae
Bradford, Mary Katherine
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Endocytosis is the process by which cells internalize extracellular and transmembrane cargo from their environment. The process requires dozens of proteins to be recruited in a specific order to the site of endocytosis on the plasma membrane. These proteins bind the cargo to be internalized, form a coat on the membrane, deform the membrane using the force of actin polymerization to create the nascent vesicle, and finally pinch off the vesicle to allow for the cargo to be trafficked within the cell. Clathrin-mediated endocytosis (CME) is the most studied endocytic mechanism; both the order of events and the proteins involved are well conserved in eukaryotic organisms. Due to quick reproduction times and ease of genetic manipulations, Saccharomyces cerevisiae is an ideal model system to gain insight into the precise mechanisms that are required to coordinate CME. The timeline of CME is separated into four steps based on the proteins that are recruited during that stage: early coat, late coat, actin polymerization, and scission/uncoating. Although much is known about what proteins are recruited and when, there are still open questions concerning how each stage transitions to the next. The scaffold protein Intersectin/Pan1 may function as a coordinator between endocytic stages due to its multiple interactions with several proteins in the early, late, and actin polymerization stages. Intersectin and Pan1 are essential for life, making research of their function technically difficult. In this dissertation, a degron allele of Pan1 (Pan1-AID) was constructed to acutely deplete cells of Pan1 protein and observe the resulting phenotypes in vivo. Using Pan1-AID, it was discovered that Pan1 is critical for strengthening interactions during three transitions in CME: early coat to late coat, actin regulators to actin machinery, and coat/actin machinery to the plasma membrane. In addition, the regions and domains that are critical for Pan1’s endocytic and essential functions have been defined. Portions of Pan1 were found that can support viability, but not endocytosis, suggesting that these functions can be separated and Pan1, like Intersectin, may have additional roles in the cell.