MOLECULAR GENETICS OF INTRACELLULAR PROTEIN TRANSPORT

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A fundamental aspect of eukaryotic cell assembly and growth is the transport of newly synthesized proteins from the cytoplasm to one of a number of chemically distinct subcellular compartments. The secretory pathway, a prominent route of intracellular protein transport, delivers both soluble and membrane-bound proteins to the cell surface. Each step in this transport process is mediated by cargo-carrying vesicles that bud from the membrane of one compartment and fuse with the membrane of the next. The yeast Saccharomyces cerevisiae has a secretory pathway much like that of mammalian cells. An important class of genes (SEC) that drive the process of vesicular protein transport have been identified through the use of mutants that disrupt the secretory process. Little is known of the precise function of most of these genes. Dr. Kaiser proposes to develop a new method to study the function of SEC gene products by determining their precise intracellular location. The yeast Pichia pastoris is similar to S. cerevisiae in many respects except that P. pastoris cells contain prominent and morphologically well defined Golgi complexes. Methods will be devised for the isolation of P. pastoris homologs of SEC genes and the localization of their products in P. pastoris cells by antibody staining and electron microscopy. In addition, Dr. Kaiser will develop genetic screens to identify new SEC genes. The products of these new genes will also be localized in P. pastoris. Finally, by using dominant negative mutations in SEC genes, a general method for expressing secretory defects in different cell types will be tested. This method will be particularly useful for studying the function of secretory genes in diploid organisms. Elucidation of the molecular mechanism of protein transport in yeast will directly contribute to our understanding of the equivalent processes in human cells and may, therefore, speed the development of therapies for diseases caused by those viruses that use the secretory pathway to exit infected cells. In addition, some of the genes identified in this work may be particular to fungal secretory pathway and could provide selective targets for therapy against fungal pathogens.