The Regulation of Endocytosis by Snf1-related Kinases in Yeast

Abstract

Cellular homeostasis, a fundamental requirement for all living organisms, is maintained in part through endocytic downregulation. Endocytosis of nutrient transporters is regulated in response to changing environmental conditions to adjust plasma membrane (PM) protein composition for optimal cell growth. Protein networks involved in cargo capture and sorting, membrane sculpting and deformation, and vesicle scission have been well-characterized, but less is known about the networks that sense extracellular cues and relay signals to trigger endocytosis of specific transporters. In Chapter 3 of this thesis, I establish that Hal kinases belong to a yeast kinase family that is orthologous with human AMPK/Snf1-related kinases, underscoring the importance of understanding the function and regulation of these evolutionarily conserved kinases. In Chapter 4, I demonstrate that loss of Hal4 and Hal5 leads to increased internalization of various nutrient transporters, including Art1-mediated endocytosis of the methionine transporter Mup1 and Art1-independent endocytosis of the uracil transporter Fur4. In Chapter 5, I find that acute inhibition of Hal5 in the absence of Hal4 triggers rapid endocytosis of these cargo, suggesting that Hal kinases function in the amino acid sensing relay upstream of the endocytic response. In Chapter 6, I report that Hal5 localizes to the cell surface, but relocalizes away from the cell surface in response to stimulation with specific nutrients, indicating that Hal5 responds to nutrient availability. Taken together, my research indicates that Hal5 contributes to cellular homeostasis by functioning as a nutrient-responsive endocytic gatekeeper, antagonizing endocytosis and promoting stability of transporters at the PM in nutrient-limiting conditions. Broadly, my research sheds light on how signaling molecules regulate endocytic trafficking to coordinate an adaptive growth response.