Abstract - Clathrin-mediated endocytosis is the main port of entry into our cells for medically relevant substances including cholesterol-laden particles and viruses such as influenza and hepatitis. By engulfing signaling receptors, this fundamental cellular process also tunes our sensitivity to the potentially pathological actions of growth factors and neuromodulators. As such, understanding how the underlying endocytic machinery is regulated promises to reveal novel mechanisms that could be harnessed to control neoplastic, neurodegenerative, cardiovascular, and viral diseases. At the heart of the endocytic process lies the AP2 clathrin adaptor complex which appears to undergo a conformational change during vesicle formation to actively couple membrane and cargo to the clathrin coat. Despite the central role of AP2, we lack critical details about how this molecular machine is regulated in vivo. To address this need, we have developed innovative tools in C. elegans that allow us to quantify AP2 activity at multiple levels and have employed deep genetic screens to identify two conserved protein families that appear to govern AP2 conformation and activity. Our goal is to illuminate how these allosteric regulators of the endocytic machinery function mechanistically. Previously it was thought that membrane phospholipids, cytosolic cargo domains, and phosphorylation by the AP2-associated kinase (AAK1) activate AP2. Our preliminary data indicate that a conserved region of the membrane-associated Fer/Cip4 Homology Domain-only (FCHo) proteins is required to promote endocytosis by converting AP2 to an active complex. We have named this functionally important domain the AP2 Activator, or APA. In Aim 1 we will test whether the APA is sufficient to induce a structural rearrangement of AP2, as well as defining the roles of membrane, cargo, and phosphorylation in that process. We will determine where the APA binds to AP2 by screening for C. elegans mutants that escape an APA anchored to mitochondria. We will evaluate the physiological significance of AP2 phosphorylation by characterizing kinase mutants. In Aim 2 we will validate our hypothesis that adaptiN-Ear-Binding Coat- Associated Proteins (NECAP)s counteract the active (open) conformation of AP2 to ensure proper recycling of adaptor complexes. We have discovered that AP2 accumulates in a hyper-open, hyper-phosphorylated state in NECAP mutants, and that NECAPs specifically bind open, phosphorylated forms of AP2. We will determine how NECAPs regulate AP2 activity and where they function within the hierarchy of AP2 modulation using in vitro and in vivo approaches. To fully understand how NECAPs function, we will determine their structure, a...