We are studying the role that clathrin-coated vesicles serve in intracellular transport in the endocytic and secretory pathways of eukaryotic cells. We previously cloned the clathrin heavy chain gene (CHC1) from Saccharomyces cerevisiae and generated a gene deletion in order to test whether clathrin is essential for viability of yeast. It was found that yeast can survive without clathrin heavy chains in some genetic backgrounds, but not in others. This difference was due to a second genetic locus which was termed suppressor of clathrin deficiency (SCD). With SCD1 allele, clathrin deficient yeast are not viable, and with the scd1 allele, cells lacking heavy chains survive. The goal of this project is to perform a molecular characterization of the SCD locus and its gene products. The SCD1 and scd1 alleles will be cloned and sequenced, and null mutations will be generated by gene disruption. Antibodies to the Scd1 protein will be made in order to identify the gene product in cell extracts and for cellular localization. These studies will allow us to elucidate the function of the Scd1 protein and to determine how the scd1 allele compensates for the lack of clathrin. Clathrin's ubiquity among eukaryotic organsims, and the complex regulation of clathrin assembly and disassembly, suggest that clathrin's function in cells is extremely important. Elucidation of the Scd gene product and its role in allowing cell survival in the absence of clathrin will provide important new insights into the biology of clathrin itself.
