Analysis of cis and trans acting endocytosis factors in Saccharomyces cerevisiae: Insights into classic and ubiquitin directed protein traffic

Protein traffic through the endocytic pathway is critical for regulating dynamic organization of the cell surface and sorting proteins to proper subcellular locations. By directing protein traffic from and to the cell surface, the endocytic pathway participates in diverse processes including nutrient uptake, down-regulation of signal transduction events, antigen presentation, and re-organization of cell surface components. Through these processes, endocytosis influences interactions with the external environment central to cell growth, differentiation, and response to extracellular pathogens.; Many cell surface proteins are internalized via clathrin-coated vesicles. Such cargo proteins are collected in nascent vesicles through interactions between sorting signals in their cytoplasmic domains and vesicle coat protein. Formation of clathrin coated vesicles relies upon an interplay between coat components, endocytic machinery, lipid modification, and the actin cytoskeleton. In the budding yeast Saccharomyces cerevisiae, adaptor complexes and dynamin do not appear to participate, while only indirect evidence suggest clathrin participates. However, a requirement for normal actin cytoskeleton dynamics and other conserved machinery suggest strong similarities between the molecular mechanisms that direct endocytosis and endosomal sorting in higher and lower eukaryotes.; The work presented here outlines analysis of cis and trans factors that direct sorting in the yeast endocytic pathway. Initially, we present discovery of a primary endocytosis signal, NPFX (1--2)D, the second of two endocytosis signals identified in yeast. We also demonstrate that Sla1p serves as the endocytosis adaptor for this signal, allowing construction of a model for cargo recognition, actin stimulation, lipid association, and clathrin binding---all general features of eukaryote endocytic vesicle formation.; We also present that mono-ubiquitin can direct rapid vacuole mediated turnover of internalized endocytic cargo in an endosome mediated protein trafficking event. We delineate the first ubiquitin binding domain which requires the Phe4 position of ubiquitin to direct interactions (others require the Ile44 position). We present preliminary evidence for non-Phe4/non-Ile44 ubiquitin binding domains. Finally, we demonstrate a trans ubiquitin requirement for efficient cell surface cargo uptake and normotopic sorting of endocytic traffic.