Genetic analysis of GPI-protein recycling via membrane

描述(由申请人提供)： 糖基磷脂酰肌醇(GPI)锚定叶酸受体(FR)结合 叶酸化合物和叶酸结合并介导它们被细胞摄取。 FR介导的转运被认为是通过一种新的富含鞘脂的物质来实现的 也包含其他GPI锚定蛋白的膜微域，以及 在细胞表面和胞内隔间之间循环，但分子 这种运输机械的部件尚未确定。我们建议 确定其产物对FR循环至关重要的基因及其相关基因 酵母系统中叶酸吸收的遗传分析。选择了系统 因为它的通用性和基于已知的膜的密切相似性 酵母和哺乳动物细胞之间的运输机制以及 酵母与哺乳动物膜结合特性的相似性 GPI锚定蛋白。我们将使用一种酵母叶酸营养缺陷菌，其中叶酸 摄取(可能通过扩散)需要非常高的(约100微米) 外源叶酸浓度。我们已经将人FR导入到这种酵母中 菌株在铜++诱导启动子的控制下，发现受体 在纳摩尔细胞外浓度下介导[~3H]叶酸摄取， 在小于0.1微米的叶酸中支持细胞生长，并使 低浓度的强效抗叶酸药物， 二氮杂四氢叶酸(DDATHF)。我们将对表达FR的酵母进行诱变 通过化学或通过转座子插入，并初步选择 能抵抗低浓度的敌敌畏。第二个更严格的复制品 平板筛选将通过以下途径缩小FR介导转运缺陷的突变株 选择需要高浓度(约100微米)的 外源叶酸可促进正常生长。运输缺陷将是 经[~3H]叶酸摄取研究证实。或者， 叶酸摄取缺陷的温度敏感突变体也将被分离出来。 FR突变体，以及FR合成或GPI修饰受损的突变体， 将不会被考虑。酵母基因组文库及其在人中的表达 图书馆将被用来弥补运输上的缺陷。酵母菌与人类 补充突变表型和酵母突变的基因将 通过DNA序列分析进行检测。对于这样鉴定的酵母基因， 将对假定的人类同源基因进行突变互补测试 表型。这些已知的和新的蛋白质在细胞内的机制作用 FR介导的运输将是未来调查的主题。这些研究 有望提供对膜的基本性质的新见解 运输过程。

DESCRIPTION (provided by applicant): The glycosyl-phosphatidylinositol (GPI)-anchored folate receptor (FR) binds folate compounds and folate conjugates and mediates their uptake by cells. FR-mediated transport is believed to occur via a novel sphingolipid-rich membrane microdomain that also contains other GPI-anchored proteins and that recycles between the cell surface and endocytic compartments, but the molecular components of this transport machinery have not been identified. We propose to identify genes whose products are essential for FR recycling and the associated folate uptake by genetic analysis in a yeast system. The system is chosen because of its versatility and based on the close similarity in known membrane transport mechanisms between yeast and mammalian cells as well as the similarity in membrane-associated characteristics of yeast and mammalian GPI-anchored proteins. We will use a yeast folate auxotroph in which folate uptake (presumably by diffusion) requires a very high (about 100 microM) exogenous folate concentration. We have introduced human FR into this yeast strain under control of a Cu++-inducible promoter and found that the receptor mediates [3H]folic acid uptake at nanomolar extracellular concentrations, supports cell growth in less than 0.1 microM folinic acid and sensitizes the cells to low concentrations of the potent antifolate drug, dideazatetrahydrofolate (DDATHF). We will mutagenize the FR expressing yeast chemically or by transposon insertion and initially select for mutants that are resistant to low concentrations of DDATHF. A second more stringent replica plating screen will narrow mutants defective in FR-mediated transport by selecting for those that require a high (about 100 microM) concentration of exogenous folinic acid for normal growth. The transport defects will be confirmed by [3H]folic acid uptake studies. Alternatively, temperature-sensitive mutants defective in folate uptake will also be isolated. FR mutants, as well as mutants with impaired FR synthesis or GPI-modification, will not be considered. A yeast genomic library and a human cDNA expression library will be used to complement the transport defects. The yeast and human genes complementing the mutant phenotypes as well as the yeast mutations will be examined by DNA sequence analysis. For the yeast genes thus identified, putative human homologs will be tested for complementation of mutant phenotypes. The mechanistic roles of such known and novel proteins in FR-mediated transport will be the subject of future investigations. The studies are expected to provide new insights of a fundamental nature into membrane transport processes.