BASIS FOR GOLGI LOCALIZATION OF GLYCOSYLTRANSFERASES

糖基转移酶高尔基体定位的基础

• 批准号: 3468914
• 负责人: KAREN J. COLLEY
• 金额: $ 9.67万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468914
• 关键词: Golgi apparatus; active sites; carbohydrate sequence; chimeric proteins; endoplasmic reticulum; glycoprotein structure; glycosyltransferase; immunoelectron microscopy; immunofluorescence technique; laboratory rabbit; laboratory rat; oligosaccharides; polymerase chain reaction; protein signal sequence; protein transport; sialyltransferases; site directed mutagenesis

The terminal Golgi glycosyltransferases are largely responsible for the wide array of oligosaccharide structures which modify proteins and lipids. Specific oligosaccharide structures are frequently required for the folding, stability and biologic activity of proteins and a variety of cell- cell interactions involved in development and disease. The types of oligosaccharide structures which modify proteins and lipids depend on both the presence of specific glycosyltransferases and the precise compartmentation of these enzymes in the cisternae of the Golgi apparatus. The molecular basis for the precise localization of these Golgi glycosyltransferases and Golgi proteins, in general, is not known. The long term goal of this proposal is to elucidate the mechanisms underlying the Golgi apparatus localization of Golgi terminal glycosyltransferases and other Golgi proteins. It is expected that general principles of Golgi apparatus protein localization will emerge from the investigation of the signals and mechanisms underlying the Golgi localization of a unique glycosyltransferase, the alpha2, 6-sialyltransferase (ST), which has a dual localization in the trans cisternae of the Golgi apparatus and trans Golgi network. Preliminary evidence suggests that the ST possesses Golgi localization signals in both the signal anchor domain and the stem region and that these regions also mediate the oligomerization of the enzyme. The focus of this research proposal will be 1) to determine which sequences in the signal anchor domain and stem region are necessary and sufficient for the localization of the ST and other Golgi proteins to the Golgi apparatus, 2) to investigate the role of oligomerization in the transport and sorting of the ST, and 3) to determine what protein and/or lipid components of the Golgi apparatus participate in ST Golgi localization. These aims will be accomplished by the biochemical analysis and immunofluorescence and immunoelectron microscopic localization of mutant and chimeric ST proteins constructed by in vitro oligonucleotide-directed mutagenesis and PCR techniques. The results of these studies will be fundamentally important for the understanding protein transport and sorting in the secretory pathway, and more specifically for the understanding of Golgi protein localization and the assembly of the Golgi apparatus. In addition, these studies will begin to evaluate the molecular basis for the localization of terminal glycosyltransferases across the Golgi stack and how this strict compartmentation of glycosyltransferases ultimately controls the structure of oligosaccharides on glycoproteins and glycolipids.

末端高尔基体糖基转移酶主要负责 多种修饰蛋白质和脂质的寡糖结构。 特定的寡糖结构经常需要 蛋白质和多种细胞的折叠、稳定性和生物活性 细胞相互作用参与发育和疾病。 的类型 修饰蛋白质和脂质的寡糖结构取决于两者 特定糖基转移酶的存在和精确的 这些酶在高尔基体池中的划分。 这些高尔基体精确定位的分子基础 一般来说，糖基转移酶和高尔基体蛋白是未知的。 这 该提案的长期目标是阐明潜在的机制 高尔基体末端糖基转移酶的高尔基体定位和 其他高尔基体蛋白。 预计高尔基体的一般原则 装置蛋白定位将从对 高尔基体定位的信号和机制 糖基转移酶，α2, 6-唾液酸转移酶 (ST)，具有双重作用 定位于高尔基体横池和高尔基体横池 网络。 初步证据表明 ST 拥有高尔基体 信号锚域和茎区域中的定位信号 并且这些区域还介导酶的寡聚化。 这 本研究提案的重点是 1) 确定哪些序列 信号锚域和茎区对于 ST 和其他高尔基体蛋白在高尔基体的定位， 2）研究低聚在运输和分选中的作用 ST 的组成，以及 3) 确定 ST 的蛋白质和/或脂质成分 高尔基体参与 ST 高尔基体定位。 这些目标将是 通过生化分析和免疫荧光法完成 突变型和嵌合型 ST 蛋白的免疫电镜定位 通过体外寡核苷酸定向诱变和 PCR 构建 技术。 这些研究的结果将具有根本性的重要意义 用于了解分泌中的蛋白质运输和分类 途径，更具体地说是为了了解高尔基体蛋白 高尔基体的定位和组装。 此外，这些 研究将开始评估定位的分子基础 穿过高尔基体堆栈的末端糖基转移酶以及这种严格的 糖基转移酶的区室最终控制结构 糖蛋白和糖脂上的寡糖。