All eukaryotic cells take up molecules from the extracellular environment through a process called endocytosis. In the best-characterized endocytic process, a self-assembling protein called clathrin forms a slightly invaginated, lattice-like structure on the inside of the plasma membrane called a clathrin-coated pit. When an extracellular molecule, such as the LDL particles in our bloodstream, binds to its cell surface receptor, the complex migrates into one of these clathrin-coated pits and is captured through interaction with clathrin adaptor proteins with the receptor. After the pit has captured a few receptors with bound cargo, it invaginates further into the cell and pinches off from the plasma membrane to form a clathrin-coated vesicle. The clathrin coat is then rapidly stripped from the clathrin-coated vesicles to allow recycling of clathrin back to the plasma membrane, and to allow fusion the naked vesicle with an endosome. The assembly and disassembly phases of the clathrin cycle must be tightly regulated to ensure an orderly progression of vesicle budding and uncoating, but how such a dynamic system is regulated in living cells is poorly understood. This project will define the in vivo function of auxilin, a key regulator of Hsp70 activity in clathrin uncoating, using the facile molecular genetics of the budding yeast (Saccharomyces cerevisiae) for a model system. Yeast auxilin (called Swa2p) is a modular protein consisting of N-terminal clathrin binding motifs and a ubiquitin binding (UBA) domain, a centrally located TPR domain that may be used for interaction with Hsp70, and a C-terminal J domain that can stimulate the Hsp70 ATPase activity. This project will define the contribution of each domain to auxilin function in living cells.
Broader Impacts. In addition to the scientific importance of understanding how clathrin dynamics is regulated, this project will continue to contribute to science education and infrastructure. Initiated through an NSF career award, the arf1 synthetic lethal screen has been performed by hundreds of undergraduate students, giving them the opportunity to participate in the discovery process of science. Moreover, undergraduate students have cloned several of the SWA genes discovered in this screen. The participation of undergraduate students will be continued to engage more students in a discovery-based research experience.
