BIOSYNTHESIS OF MEMBRANE PROTEIN GLYCOLIPID ANCHORS

糖基磷脂酰肌醇(GGIs)是一种复杂的糖脂 在真核生物中普遍存在。这些脂类被发现 共价连接到细胞表面糖蛋白，并被识别为 是一种重要的替代蛋白质锚定机制 细胞膜。GPI锚定蛋白似乎是 膜结构域在功能上很重要 细胞内膜交通和跨膜信号转导。如果 GPI向蛋白质的转移被阻止，蛋白质不在 细胞表面，随之而来的相关细胞功能的丧失。 这项延续提案的目的是探讨 GPI在哺乳动物细胞和细胞内的组装和细胞动力学 原生动物，长期目标是获得对 GPI在细胞功能中的作用。具体目标是 GPI及其共价键的细胞内转运研究 GPI对蛋白质的修饰。GPI是在 内质网(ER)和非蛋白连接的GPI是 已知可运输到质膜上。我们建议 这种转运是通过蛋白质辅助的转移通过 胞浆，导致GPI分布到细胞质中 任何可接受的细胞膜的表面。我们打算继续推进这一进程 通过研究亚细胞的分布和运输提出假说 哺乳动物体内一系列代谢性标记的GPI结构 细胞，通过对GPI跨双层分布的分析 质膜(使用水泡性口炎病毒作为 从拓扑上正确地制备质膜)和通过重新 在无单元格系统中创建GPI传输，目的是 分离与运输相关的胞质因子。总体目标 这些研究中的一项是获得GPI的分子描述 哺乳动物细胞中的运输，目的是照亮电流 细胞内脂质转运和膜GPI的概念 信号转导通路。GPI与蛋白质发生连接 通过内质网中的一种新的转胺化反应，需要 内质网膜蛋白的参与。转氨酶 活动的特征有限，但推定的 与酶活性相对应的多肽复合体 仍有待分离。我们打算鉴定转氨酶和 其他参与GPI附着的蛋白质通过使用非细胞 蛋白质转位-GPI-锚定系统，与 化学和光交联，以及生物化学分级。 同时，我们还打算探索一种蛋白质易位 转氨酶活性的独立测定，可用于 活性的提纯。预计这两种方法都将 发现与GPI锚定有关的候选蛋白。

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.