Endocytosis is the process by which eukaryotic cells take up materials from their immediate surroundings and internalize their own surface membranes by invaginating small patches of the plasma membrane and then pinching these invaginations off. The endocytotic process is complex and highly regulated, and often involves receptor-mediated uptake of biologically important external molecules. Work in the yeast Saccharomyces cerevisiae has identified a mode of endocytosis in which ubiquitin plays a central role. Ubiquitin is a small (76 amino acids) polypeptide that has been well characterized in terms of its role in marking or tagging proteins destined for proteolytic turnover. However, the recent work of Dr. Davis and others has demonstrated that ubiquitin also plays another important role in the life of the cell, in this case marking or tagging membrane proteins for endocytosis. The covalent attachment of ubiquitin to selected plasma membrane substrates provides the sorting determinant that triggers uptake. Though initially characterized in yeast, ubiquitin-dependent endocytosis is not yeast-limited: several mammalian plasma membrane proteins have long been known to be ubiquitinated and recent evidence links ubiquitination to endocytosis. While ubiquitin clearly provides the initiating signal, nothing is presently known about how the attached ubiquitin is recognized or how the plasma membrane is packaged for uptake. Protein(s) must bind to the attached ubiquitin signal to "decode" it, and then other endocytic factors and coat proteins are expected to act downstream, packaging the endocytic substrate for uptake. This this project will identify and characterize the yeast protein(s) that serves as the ubiquitin recognition factor for uptake. Identification of this protein will potentiate the identification of the downstream components of the endocytic apparatus, the proteins, including perhaps the coat proteins, that serve to package the endocytic substrate into the endocytic complex. With key components identified, a biochemical analysis will follow how the uptake complex is assembled. Beyond this project, the ultimate goal is to build a molecular picture for ubiquitin-dependent uptake as rich in detail as the current picture for the clathrin-mediated uptake process. As the ubiquitinated plasma membrane substrate ultimately is delivered to the vacuole (yeast lysosome) for degradation, the attached ubiquitin also may provide a sorting determinant for downstream, endosomal sorting steps. Indeed, preliminary evidence is consistent with such a role. Another goal of this project, therefore, is the identification and characterization of such late-acting ubiquitin recognition factors. A final aspect of this work will be to expand upon an initial discovery of receptor recycling in yeast. This recycling, associated with the ligand-dependent uptake of the yeast a-factor pheromone receptor, is the first direct demonstration of receptor recycling in yeast. Again for this process, ubiquitin may provide the key sorting determinant, directing transport to the vacuole for degradation, versus the alternative -- recycling of receptor back to the cell surface for additional rounds of endocytosis.