METABOLISM AND TRANSPORT OF MAMMALIAN GPIS

本研究的重点是哺乳动物糖基磷脂酰肌醇（gpi）的代谢和运输，这种游离糖脂（即非蛋白连接）可能作为蛋白质的膜锚点。在临床疾病阵发性夜间血红蛋白尿的遗传缺陷中，GPI的合成在第一步被阻断。游离gpi在内质网中合成，在内质网中发生向蛋白质的转移，但它们也能够迁移到包括质膜（PM）在内的其他细胞内区室。PM和高尔基体可能含有富含GPIs的耐Triton X-100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，并提出用内质网标记物作为免疫纯化目标来表征ER-TIM的组成。这种类型的膜组织对GPI代谢的影响将在第二个目标中进行研究。我们的工作假设是，内质网中TIM中游离的GPIs的代谢活性远低于TIM外的游离GPIs。将在完整细胞和内质网膜中比较TIM内外游离GPIs的周转率。此外，还将研究从积累GPI的细胞中提取的TIM和非TIM ER膜中提取的GlcN-（酰基）PI的体外甘露糖基化效率。第三个目的是研究GPIs从急诊室转运到PM的机制。已知的囊泡交通阻阻剂对放射性标记gpi运动的影响将在完整的和通透化的细胞中进行测试，并在一个重组的体外系统中进行测试，该系统由纯化的内质网膜组成，其中标记的gpi作为供体囊泡，纯化的PM/高尔基体作为受体膜。我们将确定外源性lyso-烷基- glcn - pi是如何被细胞吸收的，以及这种GPI前体在GPI锚定途径中在哪些细胞内区室中进一步代谢。在外源lyso-alkyl-GlcN-PI代谢过程中发生的头基交换活动也将被表征。这些研究将更好地理解区隔化对GPI通路调控的作用。

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.