METABOLISM AND TRANSPORT OF MAMMALIAN GPIS

这项建议侧重于哺乳动物糖基磷脂酰肌醇(GPI)的代谢和运输，GPI是一种可作为蛋白质膜锚的游离糖脂(即非蛋白质连接的)。在临床疾病阵发性睡眠性血红蛋白尿症中，GPI的合成在第一步被基因缺陷所阻止。游离的GPI是在内质网中合成的，在那里发生了向蛋白质的转移，但它们也能够迁移到其他细胞内隔室，包括质膜(PM)。PM和高尔基体可能含有富含GPI的抗Triton X-100的膜结构域。第一个目的是研究内质网(ER)中是否存在这些Triton不溶性膜(TIM)，并建议以ER标志物作为免疫纯化的靶标来表征ER-TIM的组成。第二个目的将研究这种膜组织对GPI代谢的影响。我们的工作假设是，与TIM外的游离GPI相比，内质网中隔离在TIM中的游离GPI在代谢上的活性要低得多。TIM内外游离GPI的周转率将在完整细胞和内质网细胞膜中进行比较。此外，体外甘露糖化GlcN-(酰基)PI在TIM和从积累GPI的细胞的ER膜的非TIM部分中的效率将被检测。第三个目标是调查从ER向PM传输GPI所涉及的机制已知的囊泡交通阻滞剂对放射性标记的GPI运动的影响将在完整和通透性的细胞中以及由含有标记的GPI作为供体囊泡和纯化的PM/Golgi组分作为受体膜的纯化ER膜组成的体外重组系统中进行测试。我们将确定外源溶烷基-GlcN-PI是如何被细胞摄取的，以及在GPI锚定途径中，这种GPI前体在细胞内的哪些隔室中进一步代谢。还将表征在外源赖氨酸烷基-GlcN-PI代谢过程中发生的头基交换活动。这些研究将有助于更好地理解部件化在GPI途径调控中所起的作用。

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.