GTPase Regulation of the Golgi Complex

Project Summary / Abstract A hallmark of eukaryotic cells is their compartmentalization. The evolution of membrane-bound organelles enabled separation, concentration, and specialization of functions critical for cell behavior. Yet this compartmentalization created challenges for cells, which must control the flow of material into and out of these organelles. Our lab's long-term goals are to elucidate how cells exert control over the flow of proteins and lipids within the endomembrane system. We seek to understand the Golgi complex, which serves as the nexus of many different trafficking pathways in eukaryotic cells. We have focused many of our investigations on the Golgi- localized GTPases, their regulators, and their effectors to determine how they govern protein trafficking through this enigmatic organelle. Research projects in our lab use multiple approaches, including protein biochemistry, live-cell imaging, in vitro reconstitution, and structural biology to discover fundamental mechanisms. Our work has resulted in significant advances in our understanding of how the function of the Golgi is regulated: we uncovered an extensive network of cross-talk interactions between GTPase pathways regulating Golgi trafficking, we established the pathways and substrates regulated by the Rab-activating TRAPP complexes, we identified new effectors of Arf GTPases, and we determined the structural and mechanistic basis for regulated activation of several Golgi GTPases of the Arf and Rab families. Our work has revealed how the structures of each of these essential regulators are significantly different, even those that share common domains or subunits, and the structures explain their function and regulation. Our work has underscored the importance of membranes as more than just locations where GTPase signaling reactions occur. Organelle membranes are platforms for hundreds of biochemical reactions and interactions in cells, so we need a fundamental biochemical understanding of how proteins behave on the surface of membranes in order to understand the mechanisms driving cell biology. There are over 170 Ras-related GTPases in human cells, and most of their signaling reactions occur on the surface of organelle membranes. Although many of these GTPases have been studied in great detail, the role of the membrane surface in controlling, regulating, and coordinating GTPase signaling reactions remains poorly understood. Our goals for the next five years are to address several key questions in the field using a multidisciplinary approach. We will use cryoEM and functional experiments to visualize and understand how interactions with organelle membranes and crosstalk regulators influence the structure and function of GTPase signaling reactions. We will determine how the activation of Arf GTPases is coupled to their insertion into membranes. We will use new screening approaches to identify new GTPase-interacting proteins and we will experimentally characterize the functions and roles of these proteins as potential regulators or effectors. Our planned work has broad significance for understanding signaling, trafficking, organelle homeostasis, and membrane biology.