Genetic analysis of GPI-protein recycling via membrane

GPI-蛋白质通过膜回收的遗传分析

• 批准号: 6440455
• 负责人: Manohar Ratnam
• 金额: $ 14.7万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6440455
• 关键词: Saccharomyces cerevisiae; auxotrophy; cell membrane; complementary DNA; flow cytometry; folate; fungal genetics; gene mutation; genetic library; glycosylphosphatidylinositols; intracellular transport; nucleic acid quantitation /detection; phenotype; receptor binding; temperature sensitive mutant; transposon /insertion element; vitamin receptor

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.

描述（由申请人提供）： 糖基 - 磷脂酰肌醇（GPI）锚定的叶酸受体（FR）结合 叶酸化合物和叶酸结合物并介导细胞的摄取。 FR介导的运输被认为是通过新颖的鞘脂富集出现的 膜微域也包含其他GPI锚定蛋白，并且 在细胞表面和内吞室之间回收，但分子 该运输机械的组件尚未确定。我们建议 识别其产品对于FR回收至关重要的基因及相关的基因 通过酵母系统中的遗传分析叶酸摄取。选择系统 由于其多功能性并基于已知膜的紧密相似性 酵母菌和哺乳动物细胞之间的运输机制以及 酵母和哺乳动物膜相关特征的相似性 GPI锚定蛋白。我们将使用酵母叶酸的合子，其中叶酸 吸收（大概是通过扩散）需要很高（约100 microm） 外源叶酸浓度。我们已经将人类fr介绍给了这个酵母 控制Cu ++ - 诱导启动子的应变，发现受体 介导[3H]在纳摩尔细胞外浓度下的叶酸摄取， 支持小于0.1 microm叶酸的细胞生长，并使其敏感 细胞到低浓度的有效抗卵形药物， dideazatetrahyrofaly（ddathf）。我们将诱变表达的酵母 化学或通过转座子插入，最初选择的突变体 对低浓度的DDATHF的抗性。第二个更严格的复制品 电镀屏幕会狭窄的突变体在FR介导的运输中有缺陷 选择需要高（约100微米）浓度的人 外源性叶酸的正常生长。运输缺陷将是 通过[3H]叶酸摄取研究证实。或者， 叶酸摄取中有缺陷的温度敏感突变体也将分离出来。 FR突变体以及FR合成或GPI修饰受损的突变体， 不会考虑。酵母基因组文库和人cDNA表达 库将用于补充运输缺陷。酵母和人类 补充突变表型以及酵母突变的基因将 通过DNA序列分析检查。对于如此鉴定的酵母基因， 假定的人类同源物将经过测试以补充突变体 表型。这种已知和新型蛋白质的机械作用 FR介导的运输将成为未来调查的主题。研究 期望将基本性质的新见解为膜 运输过程。