Membranous organelles in eukaryotic cells have unique phospholipid compositions which cannot be accounted for by organelle specific lipid synthesis. Understanding the physiological significance of these distinct lipid compositions and the molecular mechanisms by which they are established and maintained is one of the most fundamental, least understood problems in cell biology. Gaining this understanding is the long term goal of this proposal. The experimental approach is based on the use of fluorescent-labeled phosphatidylethanolamine to identify the genes and characterize the gene products required for proper sorting and trafficking of this phospholipid in the yeast, Saccharomyces cerevisiae. The tools of cell biology and biochemistry will be combined with the powerful and facile genetic approaches available in yeast to achieve the overall goal of understanding how and why phosphatidylethanolamine is sorted and trafficked between organellar membranes. The first specific aim is to isolate mutants that are defective in trafficking of phosphatidylethanolamine (tpe mutants) by enrichment following dye-sensitized photokilling. Mutagenized cells will be incubated with fluorescent-labeled phosphatidylethanolamine and irradiated with light that excites the fluorophore, thereby killing normal cells having internalized the fluorescent lipid and enriching for defective (mutant) cells. This technique has been used to identify two tpe mutants and will be used to identify additional genes required for proper phosphatidylethanolamine sorting and trafficking. The second specific aim is to determine the function of the tpe gene products and the significance of these functions to normal cellular physiology. This will be accomplished by cloning and sequencing the TPE genes to identify functional domains and homologies with other proteins and by the use of in vitro and in situ assays to test directly putative functions. The proteins that catalyze and regulate a wide range of cellular functions in yeast have a remarkable degree of homology with their mammalian counterparts. Given this structural homology and the observed conservation of molecular mechanisms for lipid uptake found in preliminary studies for this proposal, new advances describing the molecular details of lipid trafficking in yeast are likely to be directly applicable to understanding lipid trafficking in mammalian cells. Furthermore, the identification of differences in essential lipid sorting pathways between yeast and mammals may provide new rationales for the development of antifungal therapies.
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