Analysis of ubiquitin-dependent protein sorting within the endocytic pathway in Saccharomyces cerevisiae

The plasma membrane protein composition of a eukaryotic cell is maintained in part through the removal of transmembrane proteins by endocytosis and delivery to the lysosome (or vacuole in yeast) for degradation. The endocytic and biosynthetic pathways converge at endosomes, where related sorting events occur for proteins arriving from either pathway before their lysosomal delivery. Saccharomyces cerevisiae has proven to be an excellent model organism for the study of fundamental cellular processes, and this complex process is no exception. The powerful genetics available in the yeast system have facilitated the identification of a large number of factors that drive protein sorting throughout the endocytic pathway. It is clear that ubiquitin plays a critical role in targeting cargoes into this degradative pathway and that this signal is recognized by a series of adaptor proteins between the cell surface and lysosome that are responsible for directing the cargo for degradation. Here we provide detailed protocols for studying the fate of cargo proteins within the endosomal system, as well as the role of putative ubiquitin-binding proteins.