GENETIC APPROACH TO LIPID TRAFFICKING IN YEAST

真核细胞的膜性细胞器具有独特的磷脂 细胞器特异性脂质不能解释的成分 合成.了解这些不同的生理意义 脂质组成和分子机制，他们是 建立和维护是一个最基本的， 理解细胞生物学中的问题。获得这种理解是 这是该提案的长期目标。实验方法基于 使用荧光标记的磷脂酰乙醇胺来鉴定基因 并表征正确分选所需的基因产物， 这种磷脂在酵母中的运输，酿酒酵母。 细胞生物学和生物化学的工具将与 强大而简便的遗传方法，可在酵母，以实现 总体目标是了解磷脂酰乙醇胺是如何以及为什么 在细胞器膜之间进行分类和运输。 第一个具体目标是分离出在以下方面有缺陷的突变体： 通过富集运输磷脂酰乙醇胺（TPE突变体） 在染料敏化的光杀死之后。将孵育诱变细胞 用荧光标记的磷脂酰乙醇胺， 激发荧光团的光，从而杀死正常细胞， 内化荧光脂质并富集缺陷型（突变型） 细胞这项技术已被用来确定两个tpe突变体， 用于识别适当表达所需的额外基因， 磷脂酰乙醇胺的分类和运输。第二个具体目标 是为了确定TPE基因产物的功能和意义， 这些功能对正常细胞生理的影响。这将是 通过克隆和测序TPE基因来完成， 功能结构域和与其他蛋白质的同源性，并通过使用在 体外和原位测定以直接测试推定的功能。 催化和调节多种细胞功能的蛋白质 在与它们哺乳动物具有显著程度同源性的酵母中 同行鉴于这种结构上的同源性和观察到的保守性， 脂质摄取的分子机制的初步研究发现， 这一建议，新的进展，描述了脂质的分子细节， 酵母贩运很可能直接适用于理解 哺乳动物细胞中的脂质运输。此外，识别 酵母和哺乳动物之间必需脂质分选途径的差异 可能为抗真菌治疗的发展提供新的理论基础。

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.