LIPID CARRIERS IN MEMBRANE GLYCOPROTEIN SYNTHESIS

膜糖蛋白合成中的脂质载体

• 批准号: 2176891
• 负责人: WILLIAM J LENNARZ
• 金额: $ 21.74万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176891
• 关键词: Saccharomyces cerevisiae; active sites; affinity chromatography; carbohydrate biosynthesis; carbohydrate transport; disulfide bond; dolichol; endoplasmic reticulum; enzyme mechanism; glycoprotein biosynthesis; glycoproteins; hexosyltransferase; isomerase; lipid transport; membrane lipids; membrane proteins; molecular cloning; oligosaccharides; protein folding; site directed mutagenesis; sugar nucleotide; sugar phosphates; temperature sensitive mutant; tissue /cell culture

The assembly of N-linked glycoproteins in the endoplasmic reticulum (ER) can be considered to occur in four stages: l) Synthesis of the long chain lipid, dolichyl phosphate (Dol-P); 2) Stepwise assembly, via cytoplasmic sugar nucleotides, of a lipid-linked oligosaccharide chain oriented toward the lumen; 3) Transfer of this oligosaccharide chain to nascent polypeptide chains; and 4) Folding and formation of disulfide bonds of the newly completed (glyco)protein in the lumen. Given the wide-spread distribution and the diversity of function of glycoproteins, it is important to understand how they are assembled in the normal and diseased state. Because much evidence points to a common mechanism of glycoprotein synthesis in yeast and higher eukaryotes, and because yeast offers great advantages in terms of genetic manipulation, the proposed studies will be carried out in the yeast S. cerevisiae. Specific Aim l involves identification of the enzymatic steps in formation of Dol-P. Despite considerable work in a variety of eukaryotic systems, the steps culminating in formation of Dol-P are not well defined. A novel screen will be used to identify temperature sensitive yeast mutants defective in synthesis of Dol-P and thereby determine the pathway of its synthesis. In addition, genes in this pathway that encode for novel enzymes will be cloned and sequenced. In Specific Aim 2 initial studies will focus on the reactions and topology of oligosaccharyl-PP-Dol synthesis. Although previously developed assays will be used to study the topology of the process, a new in vivo genetic approach will also be taken. Thus, a collection of temperature sensitive strains will be screened for mutations in the oligosaccharyl-PP-Dol pathway. If defects in novel reactions in the pathway are detected, the genes will be cloned, sequenced and characterized. However, a major objective will be to identify mutants in the pathway that are defective in either sugar nucleotide transporters or saccharide-lipid translocators. If such a defect is identified the gene will be cloned, and the role of the gene product in transport or translocation will be studied in vivo and in vitro. Specific Aim 3 entails studies on the role of protein disulfide isomerase (PDI) in protein folding and disulfide bond formation in the ER. A key objective will determine the molecular basis for the finding that PDI expression is essential for yeast viability. First, the possible involvement of the Cys residues in PDI will be tested by mutating them. Second, site-directed mutations will be carried out in an acidic domain of yeast PDI that has homology with a domain in rat PDI that binds peptides, and is postulated to be the site for binding of polypeptide chains. In vivo experiments will be carried out to distinguish between two models for PDI binding: Transient interaction with nascent polypeptides versus formation of a stable complex with a mature ER protein.

内质网中N连接糖蛋白的组装（ER） 可以认为在四个阶段中发生：l）长链的合成 脂质，多甲基磷酸盐（DOL-P）； 2）逐步组装，通过细胞质 脂质连接寡糖链的糖核苷酸 朝向管腔； 3）将此寡糖链转移到新生 多肽链； 4）折叠和形成的二硫键 腔内新近完成的（Glyco）蛋白。给定广泛的 分布和糖蛋白功能的多样性，这是 重要的是要了解它们如何在正常和患病中组装 状态。因为很多证据表明糖蛋白的常见机制 酵母和更高的真核生物的合成，并且因为酵母提供了很棒的 在遗传操作方面具有优势，拟议的研究将 在酵母菌酿酒酵母中进行。 特定目标涉及识别酶促步骤 DOL-P的形成。尽管在各种真核生物中都有大量工作 系统，最终形成DOL-P的步骤不好 定义。新颖的屏幕将用于识别温度敏感 酵母突变体在合成DOL-P中有缺陷，从而确定 其合成的途径。另外，该途径中的基因编码 对于新型酶，将被克隆和测序。 在特定目标中2初步研究将重点放在反应上， 寡聚核酸-PP-DOL合成的拓扑。虽然以前 开发的测定将用于研究该过程的拓扑，这是一个新的 还将采用体内遗传方法。因此， 温度敏感菌株将在 寡素charyl-pp-dol途径。如果新反应中的缺陷 检测到途径，基因将被克隆，测序，并且 特征。但是，一个主要目标是确定 两种糖核苷酸转运蛋白中有缺陷的途径 或糖脂流动器。如果确定了这样的缺陷 基因将被克隆，以及基因产物在运输或 易位将在体内和体外研究。 特定目的3需要研究蛋白质二硫化物异构酶的作用 （PDI）在ER中的蛋白质折叠和二硫键形成中。钥匙 目标将确定PDI发现的分子基础 表达对于酵母生存能力至关重要。首先，可能 CYS残基参与PDI将通过突变来测试它们。 其次，将在酸性结构域中进​​行定点突变 与大鼠PDI中有同源性的酵母PDI的结合 肽，并被认为是多肽结合的位点 链。将进行体内实验以区分 PDI结合的两个模型：与新生的瞬态相互作用 多肽与成熟ER的稳定复合物的形成 蛋白质。