Molecular Genetics of Intracellular Protein Transfer

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DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) is the compartment where membrane and secretory proteins are modified, folded, and assembled. A key step in the folding of most extracellular proteins includes formation of disulfide bonds, which can add stability to folded polypeptides and can link together subunits of protein complexes. Previous work on this project has delineated the core pathway for protein disulfide bond formation in eukaryotic cells in which disulfide bonds generated by an oxidase, Ero1p, are then transferred to protein disulfide isomerase (PDI), which in turn transfers a disulfide bond to substrate proteins. Through a combination of biochemical, structural, and genetic experiments in the yeast S. cerevisiae we have gained fundamental insight into the molecular mechanisms that underlie each of the steps in this pathway. In this application we now focus on understanding how the protein oxidation pathway is integrated into the redox biochemistry of the cell. Aims of the proposal include: understanding how Pdi1p and related proteins regulate Ero1p activity, investigation of a novel block in ER translocation instigated by hyperactive Ero1p activity, and elucidation of a new electron transport chain in the ER coupled to disulfide bon formation. PUBLIC HEALTH RELEVANCE: In S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to uncover the genes and proteins responsible protein folding in the ER. A detailed understanding of these pathways in S. cerevisiae will make it possible to understand parallel processes in mammalian cells, opening the way to diagnose dysfunctional folding in the ER of mammalian cells and possible detrimental effects of reactive oxygen species generated by the disulfide bond formation pathway.