BASIS FOR GOLGI LOCALIZATION OF GLYCOSYLTRANSFERASES

末端高尔基糖基转移酶主要负责 种类繁多的寡糖结构，可以修饰蛋白质和脂类。 特定的寡糖结构是经常需要的 蛋白质和各种细胞的折叠、稳定性和生物活性- 参与发育和疾病的细胞相互作用。这些类型的 修饰蛋白质和脂类的低聚糖结构依赖于两者 特异性糖基转移酶的存在和精确的 高尔基体脑池中这些酶的区隔。 这些高尔基体精确定位的分子基础 糖基转移酶和高尔基体蛋白一般不为人所知。这个 这项提议的长期目标是阐明潜在的机制 高尔基体对高尔基体末端糖基转移酶的定位 其他高尔基蛋白。预计高尔基的一般原则 仪器蛋白质定位将在研究中出现 独一无二的高尔基体局部化的信号和机制 糖基转移酶，α2，6-唾液酸基转移酶(ST)，具有双重结构 高尔基体和跨高尔基体池的定位 网络。初步证据表明，ST拥有高尔基人 信号锚域和茎区域中的定位信号 这些区域也参与了酶的寡聚化。这个 本研究建议的重点将是1)确定哪些序列 信号锚定结构域和主干区域对于 ST和其他高尔基蛋白在高尔基体的定位， 2)研究齐聚在运输和分选中的作用 以及3)确定ST的哪些蛋白质和/或脂肪成分 高尔基仪器参与圣高尔基本地化。这些目标将是 由生化分析和免疫荧光完成 突变型和嵌合型ST蛋白的免疫电子显微镜定位 体外寡核苷酸定向突变和聚合酶链式反应构建 技巧。这些研究的结果将是至关重要的。 为了了解蛋白质在分泌物中的运输和分类 途径，更具体地说是为了理解高尔基体蛋白 高尔基仪器的国产化和组装。此外，这些 研究将开始评估定位的分子基础。 高尔基体末端糖基转移酶及其严格程度 糖基转移酶的区隔最终控制着结构 糖蛋白和糖脂上的低聚糖。

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.