The compartmental organization of eukaryotic cells relies on the accurate distribution of proteins to different compartments and on retention of proteins within each compartment. The ability to sort proteins with different destinations is a key feature of the transport machinery which governs protein distribution and retention. The molecular basis of this selectivity is the focus of this proposal. Studies of yeast strains carrying mutations in the clathrin heavy chain subunit revealed that clathrin participates in selective protein transport processes at the plasma membrane and the Golgi complex: endocytosis of the mating pheromone receptors, sorting of vacuolar precursors from the Golgi complex to vacuoles, and a previously unrecognized role in localization of membrane proteins to a late Golgi compartment. Additional proteins have been identified which function in Golgi membrane protein localization and a collection of mutants with defects in this process have been isolated. These studies form the foundation for a combination of genetic, cell biology and biochemical approaches to investigate three aspects of selective protein transport: 1) the mechanism of Golgi membrane protein localization; 2) the role played by clathrin-associated proteins in clathrin-dependent protein transport processes; 3) the mechanism of receptor internalization during receptor-mediated endocytosis of mating peptide pheromones. Mutants (tcs) with defects in Golgi membrane protein localization will be used to clone wild-type versions of genes whose protein products participate in the localization process. The localization of TCS-encoded proteins will be determined in wild-type and mutant strains to explore interrelationships between the Tcs proteins and proteins involved in clathrin-coated vesicle formation. To define the function of clathrin coats in Golgi membrane protein localization, gene disruptions will be generated to eliminate all subunits of a clathrin-associated protein (AP-l) complex which is postulated to play a central role in the formation of clathrin-coated vesicles at the Golgi complex. Genetic strategies will be employed to identify functionally redundant AP subunits and other proteins which interact with AP-l. Finally, a novel class of endocytosis targeting signals will be used as probes to isolate proteins responsible for selective packaging of receptors into nascent endocytic vesicles. Together, these approaches will identify previously unrecognized components of the selective protein transport machinery and address the specific roles played by each identified participant.
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