We are interested in elucidating the mechanisms by which proteins are sorted to the lysosome-like vacuole in the budding yeast Saccharomyces cerevisiae. Genetic selections have resulted in the identification of over forty genes (VPS) that are involved in vesicle-mediated transport of proteins from the Golgi to the yeast vacuole. The product of one of the genes implicated in this sorting pathway, Vps21p, is a member of the Rab family of small GTPases. Rab proteins have been implicated at most vesicle trafficking steps in eukaryotic cells. Despite their prevalence, very little is known about their actual function. To better understand the Rab cycle and the mechanisms of donor membrane targeting of each Rab, I plan to isolate the Vps21p-specific GDI displacement factor (GDF). This protein releases prenylated Vps21p from GDI at the correct donor membrane after one round of Rab-mediated targeting to allow Vps21p to function in another cycle of vesicle targeting. Also, I will use yeast genetic screens to identify modulators of the Vps21p- specific exchange factor, Vps9p. Finally, I will map the interaction sites between Vps21p and Vps9p. This set of experiments will help us to understand the Vps21p-Vps9p interaction and may help elucidate the mechanism of Vps9p stimulated nucleotide exchange on Vps21p. These experiments will further characterize the mechanisms of Rab mediated protein sorting, will aid us in our understanding of how Rab proteins are specifically targeted to the correct membrane, and will explain the nature of the specificity of each Rab effector.
