UC Berkeley

K28 is an A/B toxin that targets yeast cells and depends on endocytosis and retrograde trafficking for toxicity. Knowledge of the specific proteins, lipids, and mechanisms required for trafficking and killing by K28 remains incomplete. In this work, over 5000 yeast mutants were screened and 365 were identified that affect K28 sensitivity. Hypersensitive mutants revealed cytoprotective pathways, including stress-activated signaling and protein degradation. Resistant mutants clustered to endocytic, lipid organization, and cell wall biogenesis pathways. Strikingly, the AP2 complex, which in metazoans links endocytic cargo to the clathrin coat, but had no assigned function in yeast, was critical for K28 toxicity. Yeast AP2 localizes to endocytic sites and has a cargo-specific function in K28 uptake. Furthermore, the AP2 complex arrives early during endocytic site formation, which is a step of endocytosis that is not well understood. My research in this area expanded our knowledge of the proteins present during the initial stages of endocytic site formation and defined their order of arrival. The roles that Ede1p (homolog of Eps15) and clathrin have in regulating early stages of endocytosis were also examined. My results show Ede1p functions early in endocytic site formation, whereas clathrin likely promotes site transition to the late coat stage. Since cargo also arrives during the early stages of endocytosis, its involvement in endocytic site regulation was investigated using a secretion mutant to deplete cargo from the cell surface. Our results are consistent with a role for cargo in regulating the transition of endocytic sites from the early to late stages. In total, this work comprehensively identified processes important for A/B toxin trafficking and killing, and analyzed the regulation of early endocytic sites.