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Phospholipid Flip across the Yeast Plasma Membrane

磷脂穿过酵母质膜翻转

基本信息

批准号：
7005232
负责人：
John Wylie Nichols
金额：
$ 0.83万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-02-01 至 2006-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): A combination of selective synthesis, degradation and transport produces a non-random phospholipid distribution across the plasma membrane of most eukaryotic cells. The long-term objectives of our research program are to understand the mechanisms by which this asymmetric phospholipid distribution is established and its significance to cellular function. Preliminary experiments demonstrated that in the yeast, S. cerevisiae, inward-directed transport (flip) of phosphatidyicholine (PtdCho), as reported by a short chain, fluorescent-labeled PtdCho (NBD-PC), is coupled to the plasma membrane, proton-electrochemical gradient and is down-regulated by nutrient deprivation and by activated drug resistance transcription factors. A classical mutagenesis approach identified a loss of function mutation in a gene that reduces NBD-PC flip by >90% and dramatically increases resistance to the toxic lysophospholipid analogue, ET-18-O-CH3. This gene is predicted to encode a membrane protein with two transmembrane domains and has no identifiable functional motifs. Although no molecular function has been identified, two additional homologues exist in the S. cerevisiae genome as well as in a wide range of metazoans including humans. The specific aims of this proposal are to test the hypothesis that the three yeast genes encode plasma membrane-localized, inward-directed, phospholipid transporters (flippases) that are regulated in response to growth state and toxic stress. Specifically these genes will be characterized by classic molecular, genetic, and biochemical analyses to determine the relationship of their expression to in vivo function, their cellular location, specific domains essential to function, interactions with other proteins, response to nutrient deprivation and drug resistance factors, and their in vitro flippase activity. In addition to providing a better understanding of the role of phospholipid membrane dynamics to cell function, these studies will likely identify the mechanism of internalization of ET-18-O-CH3, and other ether lipid drugs, that have anti-fungal. anti-protozoal, and anti-neoplastic activity. Understanding the mechanism by which these drugs are internalized may lead to the discovery of new drugs of this class that are internalized more efficiently and are less susceptible to the development of resistance.

描述（由申请人提供）：选择性合成，降解和运输产生非随机磷脂分布穿过大多数真核细胞的质膜。远景目标我们的研究计划的一个重要部分是了解这种机制，不对称磷脂分布的建立及其意义细胞功能初步实验表明，在酵母中，S. 酿酒酵母，磷脂酰胆碱（PtdCho）的内向转运（翻转），如短链所报道的，荧光标记的PtdCho（NBD-PC），到质膜，质子电化学梯度和下调通过营养剥夺和激活的耐药转录因子。一种经典的诱变方法鉴定了一种在一个突变体中的功能缺失突变。基因，减少NBD-PC翻转>90%，并显着增加对毒性溶血磷脂类似物ET-18-O-CH 3。据预测，该基因编码具有两个跨膜结构域的膜蛋白，可识别的功能基序。虽然没有分子功能，鉴定，另外两个同源物存在于S.酿酒酵母基因组包括人类在内的多种后生动物中也有。具体目标是我们的建议是测试这三个酵母基因编码血浆的假设，膜定位，内向，磷脂转运蛋白（翻转酶），受生长状态和毒性应激的调节。特别是这些基因将通过经典的分子、遗传和生物化学方法来表征，分析以确定它们的表达与体内功能的关系，它们的细胞位置、功能所必需的特定结构域、相互作用与其他蛋白质，对营养剥夺和耐药性的反应因子及其体外翻转酶活性。除了提供更好地理解磷脂膜动力学对细胞的作用功能，这些研究将可能确定内在化的机制 ET-18-O-CH 3和其他醚脂质药物，具有抗真菌作用。抗原生动物和抗肿瘤活性。通过以下方式了解机制这些药物的内在化可能会导致发现新的药物，这类人更有效地内化，抵抗力的发展