Identification and characterization of factors involved in the AP-3 dependent protein transport pathway of Saccharomyces cerevisiae.

The AP-3 pathway in yeast mediates cargo selective protein transport from the Golgi apparatus directly to the lysosome-like vacuole. AP-3 is highly conserved throughout evolution and is present in yeast and humans alike. Mutations of AP-3 in humans and mice can cause the genetic disorder, Hermansky-Pudlak syndrome, which is a disease characterized by prolonged bleeding times and oculocutaneous albinism. Both of these symptoms are the direct result of a failure to form lysosome related organelles, indicating the importance of AP-3 to biogenesis of these subcellular compartments. Because of the great degree of homology between yeast AP-3 and mammalian AP-3, studying AP-3 in yeast may lead to insights into the function of mammalian AP-3. Here I present the work performed pursuing a more detailed understanding of the AP-3 pathway. In Chapter 2, I present the work of my colleagues and me who developed a technique to rapidly screen through a library of deletion strains to identify mutations that have perturbed protein transport pathways. In Chapter 3, I present my efforts to utilize this screening technique to identify gene deletions that affect the AP-3 pathway. In total, 49 single gene deletion strains were found to alter the transport of ALP, a protein that is selectively sorted into the AP-3 pathway. Many of these genes have well characterized roles in protein transport steps from vesicle formation at the donor membrane to vesicle fusion at the target membrane. The AP-3 subunits and YCK3 were the only genes identified to have a specific defect in AP-3 mediated transport. The AP-3 pathway mediates cargo-selective transport with few specific factors and, instead, utilizes protein machinery that is shared among various intracellular transport pathways to function.