The long term objective is to gain insight into the mechanisms by which intracellula sorting and traffic of membranes and membrane-associated components are controlled within cells. Many proteins are localized in specific regions or organelles and movement to and from these regions within cells requires the budding, transport and fusion of membrane vesicles. The focus concerns the role that clathrin and clathrin coated vesicles (CV) serve in these processes. This will be examined by a biochemical, cellular and molecular genetic dissection of the clathrin system in S. cerevisiae. The complete yeast clathrin heavy chain gene will be cloned, using segments of the gene that have already been isolated, and the DNA sequence determined. The phenotype of cells lacking clathrin heavy chains will be assessed by deleting the gene to determine whether clathrin is an essential gene product and/or whether clathrin is required for cell processes, such as endocytosis, secretion, vacuole formation, mating and sporulation. The effect of overexpression of clathrin in cells will be studied. This is of interest because clathrin is a structural protein which assembles with other proteins in the initiation of specific membrane vesiculation, and the coordinate synthesis and stoichiometry of these molecules in cells may be required to maintain functional clathrin activity. Point mutations in the clathrin gene will be isolated to extend the analysis of the clathrin system. It will be possible to define specific amino acid sequednces in the clathrin heavy chain which are essential for coated vesicle formation and inteaction with clathrin associated proteins. Extragenic suppressors of these clahtrin heavy chain mutants will be isolated, which will help to identify gene products that physically interact with clathrin or proteins that may substitute for clathrin. Molelcular genetic studies will be complemented by further biochemical characterization. A battery of monoclonal antibodies that bind to yeast clathrin heavy chains will be studied to define structural and functional domains on clathrin. These antibodies will also be used to identify proteins that co-assemble with clathrin, to determine the ratio of clathrin in CV (or cages) to unassembled forms in cells and to aid in the characteriztion of mutant clathrin heavy chains.