Phospholipid Flip across the Yeast Plasma Membrane

描述(申请人提供)：选择性合成、降解和运输的组合产生非随机分布的磷脂 穿过大多数真核细胞的质膜。长期目标 我们的研究计划的一部分是理解这种 建立了磷脂的不对称分布，并对其意义进行了研究 细胞功能。初步实验证明，在酵母中，S. Cerevisiae，磷脂酰胆碱(PtdCho)的内向转运(FliP)， 据短链报道，荧光标记的PtdCho(NBD-PC)是偶联的 到质膜，质子-电化学梯度被下调 通过营养剥夺和激活耐药转录因子。 一种经典的突变方法发现了一种功能缺失的突变 将NBD-PC翻转减少90%并显著增强对 有毒的溶血磷脂类似物，ET-18-O-CH3。这一基因被预测为 编码具有两个跨膜结构域的膜蛋白，并且没有 可识别的功能主题。尽管目前还没有分子功能 经鉴定，在酿酒酵母基因组中还存在另外两个同源物 就像在包括人类在内的各种后生动物中一样。这样做的具体目的是 建议对三种酵母基因编码血浆的假设进行检验 膜定位的、内向的、磷脂转运蛋白(Flippase) 是对生长状态和有毒压力的反应而调节的。特别是这些 基因将以经典的分子、遗传和生化为特征 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、功能所必需的特定区域、相互作用 与其他蛋白质一起，对营养剥夺和耐药性的反应 因子，以及它们的体外翻转酶活性。除了提供 更好地理解磷脂膜动力学对细胞的作用 功能，这些研究可能会确定内在化的机制 ET-18-O-CH3和其他具有抗真菌作用的乙醚类药物。 抗原虫和抗肿瘤活性。通过以下方式了解该机制 这些药物被内化可能会导致发现新的药物 这个类内部化得更有效，更不容易受到 抗药性的发展。

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.