BIOSYNTHESIS OF MEMBRANE PROTEIN GLYCOLIPID ANCHORS

糖基磷脂酰肌醇（GPIs）是复杂的糖脂， 在真核生物中普遍存在。 这些脂质被发现 与细胞表面糖蛋白共价连接并被识别， 是锚定蛋白质的重要替代机制， 细胞膜 GPI锚定蛋白似乎是 膜结构域在功能上是重要的， 细胞内膜运输和跨膜信号传导。 如果 GPI向蛋白质的转移被阻断，蛋白质不表达。 细胞表面，从而丧失相关的细胞功能。 这一延续建议的目的是探讨 GPIs在哺乳动物细胞中的组装和细胞动力学， 原生动物，其长期目标是获得对 GPIs在细胞功能中的作用 具体目标是 研究GPIs的细胞内转运和共价结合， 通过GPI修饰蛋白质。 GPIs是在 内质网（ER）和非蛋白质连接的GPIs是 已知被运输到质膜。 我们提出 这种转运是通过蛋白质辅助的转运发生的， 细胞质，导致GPIs分布到细胞质 任何接受细胞膜的表面。 我们打算继续调查 假设通过研究亚细胞分布和运输 哺乳动物中一系列代谢标记的GPI结构 细胞，通过分析GPIs的跨双层分布， 质膜（使用水泡性口炎病毒作为 质膜的拓扑正确制备）和通过重新- 在无细胞系统中创建GPI运输，目的是 分离转运相关的胞质因子。 总体目标 这些研究的目的是获得GPI的分子描述 哺乳动物细胞中的运输，目的是阐明电流 细胞内脂质转运和膜GPIs的概念， 信号转导途径 GPI与蛋白质的连接发生 通过ER中的新型转酰胺化反应， ER膜蛋白的参与。 转酰胺酶 活动的特点是在有限的范围内，但推定的 对应于酶活性的多肽复合物 仍然是孤立的。 我们打算鉴定转酰胺酶， 通过使用无细胞技术， 蛋白质移位-GPI-锚定系统，结合 化学和光交联以及生化分离。 与此同时，我们还打算探索蛋白质易位 用于转酰胺酶活性的独立测定， 活性的纯化。 预计这两种方法都将 发现参与GPI锚定的候选蛋白。

Glycosylphosphatidylinositols (GPIs)are complex glycolipids that are ubiquitous in eukaryotes. These lipids were discovered covalently linked to cell-surface glycoproteins and recognized to be an important alternative mechanism for anchoring proteins to cell membranes. GPI-anchored proteins appear to be markers of membrane structural domains that are functionally important in intracellular membrane traffic and transmembrane signaling. If GPI transfer to protein is blocked, the protein is not expressed at the cell surface with consequent loss of the relevant cell function. The aim of this continuation proposal is to explore aspects of the assembly and cellular dynamics of GPIs in mammalian cells and protozoa, with the long term goal of acquiring an appreciation of the role of GPIs in cell function. The specific objectives are to study the intracellular transport of GPIs and the covalent modification of proteins by GPI. GPIs are synthesized in the endoplasmic reticulum (ER), and non-protein-linked GPIs are known to be transported to the plasma membrane. We propose that transport occurs by protein-assisted transfer through the cytosol, resulting in the distribution of GPIs to the cytoplasmic face of any receptive cellular membrane. We intend to pursue this hypothesis by studying the sub-cellular distribution and transport of a range of metabolically labeled GPI structures in mammalian cells, by analyses of the transbilayer distribution of GPIs at the plasma membrane (using vesicular stomatitis virus as a topologically correct preparation of plasma membrane) and by re- creating GPI transport in a cell-free system with the aim of isolating transport-relevant cytosolic factors. The overall objective of these studies is to obtain a molecular description of GPI transport in mammalian cells, with the aim of illuminating current notions of intracellular lipid transport and membrane GPIs in signal transduction pathways. GPI attachment to protein occurs via a novel tansamidation reaction in the ER and requires the participation of membrane proteins of the ER. The transamidase activity has been characterized to a limited extent but the putative polypeptide complex corresponding to the enzymatic activity remains to be isolated. We intend to identify the transamidase and other proteins involved in GPI attachment by using cell-free protein translocation-GPI-anchoring systems, in conjunction with chemical and photo crosslinking, and biochemical fractionation. In parallel, we also intend to explore a protein translocation independent assay for transamidase activity that may be used for purification of the activity. Both approaches are expected to uncover candidate proteins involved in GPI anchoring.