Regulation of Traffic into and out of the Yeast Endosome by the VPS9P Cue Domain and the VPS5P Domain

The presence of membrane bound compartments in eukaryotic cells enables the generation of discrete environments in which distinct and sometimes competing chemical reactions occur. The mammalian lysosome represents an example of this principle. The lysosome is an acidic, hydrolase-rich compartment that functions in macromolecular degradation. The delivery of material to the lysosome both from biosynthetic and endocytic pathways is a highly regulated process, and defects in the lysosomal trafficking system have been linked to congenital diseases including mucolipidosis type II (I-cell disease). An analogous trafficking system functions in the fungi Saccharomyces cerevisiae to deliver biosynthetic and endocytic cargo to the yeast vacuole. Genetic and biochemical analyses of the yeast vacuolar protein sorting pathway have defined the steps in this process and identified more than 40 gene products involved. Soluble vacuolar hydrolases are diverted from the secretory pathway through interaction with a vacuolar protein sorting receptor in the trans-Golgi compartment. This receptor then facilitates transport to the endosome where the biosynthetic and endocytic pathways coincide. The receptor is recycled back to the Golgi while the vacuolar hydrolases and endocytic material are conveyed to their ultimate destination. I have been interested in two aspects of this pathway in yeast, focusing on traffic into and out of the endosome. The first question addressed is the identification of the cytosolic components that mediate receptor recycling (Chapters 2 and 3). The Sorting Nexin-1 homolog Vps5p is demonstrated to form a complex with Vps17p to mediate this process. Vps5p and Vps17p also interact with Vps26p, Vps29p and Vps35p and the lipid phosphatidylinositol-3-phosphate to recycle the receptor. The significance of these protein-protein interactions and the lipid-protein interaction in Vps5p function is examined. The second question addressed is the regulation of traffic into the endosome by modulators of the guanine nucleotide exchange factor Vps9p (Chapters 4 and 5). Ubiquitin is identified as one such regulator, and the Vps9p CUE domain is demonstrated to be a new ubiquitin binding motif. The mechanism by which the CUE domain binds ubiquitin is addressed, and the functional relevance of Vps9p ubiquitin binding and ubiquitylation are examined in vivo.