Dr. Nothwehr's laboratory studies a protein called Ste13p that carries out its function in a membrane-enclosed compartment within the cell, the trans-Golgi network (TGN). An important role of Ste13p is that it modifies mating pheromone as the pheromone is secreted from the cell; without this modification the secreted pheromone is not functional. Ste13p does not simply stay in the TGN, but is constantly being transported between the TGN and two other compartments, the early endosome (EE) and late endosome (LE). The PI's interests lie in deciphering the mechanism by which Ste13p moves between these compartments in an orderly fashion and avoids being transported to other destinations. Membrane transport typically occurs by the formation of vesicles, small compartments that pinch off from larger compartments such as the EE. Coat protein assemblies catalyze the formation of vesicles and determine which cargo proteins enter the vesicle. The PI's lab has shown that a coat called clathrin/AP-1 associates with Ste13p and the data suggest that this interaction is part of the mechanism by which Ste13p is recruited into EE-derived vesicles for transport back to the TGN. The project will study the interaction between these proteins at the atomic level to precisely define how the interaction takes place. The PI's lab also identified a specific phosphorylation of Ste13p that dictates whether Ste13p moves from the EE to the LE or from the EE to the TGN. The project will identify the enzyme that phosphorylates Ste13p and then assess the conditions that determine phosphorylation. Finally, the project will determine how phosphorylation affects the trafficking of Ste13p. The PI's hypothesis is that the presence of the phosphate group on Ste13p affects its interaction with other proteins in addition to clathrin/AP-1. Understanding how proteins are transported between membrane-enclosed compartments and are sorted has relevance to many important processes in cells, including how cells respond to instructions from other cells. The broader impact of this project will be in the training of graduate and undergraduate students, particularly in providing support for participation of individuals from under-represented groups. The PI will take advantage of two programs in place at the University of Missouri-Columbia for increasing minroity participation at the graduate level, the Louis Stokes Alliance for Minority Participation and Alliances for Graduate Education and the Professoriate. In addition, students will be recruited from the Exposure to Research for Science Students program which is designed to foster participation in research at the undergraduate level for minority and first-generation college students.
