METABOLISM AND TRANSPORT OF MAMMALIAN GPIS

哺乳动物 GPIS 的代谢和运输

• 批准号: 6628552
• 负责人: DANIEL SEVLEVER
• 金额: $ 11.13万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628552
• 关键词: Mammalia; cell membrane; endoplasmic reticulum; glycosylphosphatidylinositols; lipid metabolism; lipid transport; membrane structure; protein purification; radiotracer; vesicle /vacuole

This proposal is focused on the metabolism and transport of mammalian glycosylphosphatidylinositols (GPIs), free glycolipids (i.e. non- protein-linked) that may serve as membrane anchors of proteins. GPI synthesis is blocked at the first step by a genetic defect in the clinical disorder paroxysmal nocturnal hemoglobinuria. Free GPIs are synthesized in the ER where transfer to protein occurs, but they are also able to migrate to other intracellular compartments including the plasma membrane (PM). The PM and probably the Golgi apparatus contain membrane domains resistant to Triton X-100 that are enriched in GPIs. The first aim investigates the presence of these Triton Insoluble Membranes (TIM) in the endoplasmic reticulum (ER) and proposes to characterize the composition of ER-TIM using ER markers as targets for immunopurification. The consequences of this type of membrane organization for GPI metabolism will be studied in the second aim. Our working hypothesis is that free GPIs sequestered in TIM in the ER are much less metabolically active than free GPIs outside TIM. Turnover rates of free GPIs within and outside TIM will be compared in intact cells and in ER membranes. In addition, the efficiency of in vitro mannosylation of GlcN-(acyl)PI in TIM and in non-TIM fractions of ER membranes from cells that accumulate this GPI will be examined. The third aim investigates the mechanisms involved in transporting GPIs from the ER to the PM. The effect of known blockers of vesicular traffic on the movement of radiolabeled GPIs will be tested in intact and permeabilized cells and in a reconstituted in vitro system consisting of purified ER membranes containing labeled GPIs as donor vesicles and purified PM/Golgi fractions as acceptor membranes. We will determine how exogenous lyso-alkyl-GlcN-PI is taken up by cells and in which intracellular compartments this GPI precursor is metabolized further in the GPI anchor pathway. A head group exchange activity that take places during metabolism of exogenous lyso-alkyl-GlcN-PI will be also characterized. These studies will provide a better understanding of the role played by compart-mentalization on the regulation of the GPI pathway.

该提案集中于哺乳动物糖基磷脂酰肌醇（GPIs）、游离糖脂（即非蛋白质连接）的代谢和转运，其可用作蛋白质的膜锚。GPI合成在第一步被临床疾病阵发性睡眠性血红蛋白尿症的遗传缺陷阻断。游离GPIs在ER中合成，在ER中发生蛋白质转移，但它们也能够迁移到其他细胞内区室，包括质膜（PM）。PM和可能的高尔基体含有富含GPIs的对Triton X-100具有抗性的膜结构域。第一个目的是研究这些Triton不溶性膜（TIM）在内质网（ER）中的存在，并提出使用ER标记物作为免疫纯化的靶点来表征ER-TIM的组成。 这种类型的膜组织GPI代谢的后果将在第二个目标进行研究。我们的工作假设是，游离GPIs螯合在TIM中的ER比TIM外的游离GPIs代谢活性低得多。将在完整细胞和ER膜中比较TIM内外游离GPIs的转换率。此外，将检查来自积累该GPI的细胞的ER膜的TIM和非TIM级分中GlcN-（酰基）PI的体外甘露糖基化的效率。第三个目标是研究GPIs从ER到PM的运输机制。将在完整和透化的细胞中以及由含有标记的GPI作为供体囊泡的纯化ER膜和含有标记的GPI作为受体膜的纯化PM/Golgi部分组成的重建体外系统中测试已知囊泡运输阻滞剂对放射性标记的GPI运动的影响。我们将确定外源性溶血-烷基-GlcN-PI是如何被细胞吸收的，以及该GPI前体在GPI锚途径中进一步代谢的细胞内隔室。 还将表征在外源性溶血-烷基-GlcN-PI代谢期间发生的头基交换活性。这些研究将提供一个更好的了解所发挥的作用，区室化的调节GPI途径。