BASIS FOR GOLGI LOCALIZATION OF GLYCOSYLTRANSFERASES

末端高尔基体糖基转移酶在很大程度上负责 修饰蛋白质和脂质的各种寡糖结构。 特定的寡糖结构经常需要用于 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。 的类型 修饰蛋白质和脂质的寡糖结构依赖于 特异性糖基转移酶的存在和精确的 这些酶在高尔基体池中的区室化。 精确定位这些高尔基体的分子基础 糖基转移酶和高尔基体蛋白的功能通常是未知的。 的 这项建议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定位， 其他高尔基体蛋白。 预计高尔基体的一般原理 器蛋白定位将出现从调查的 信号和机制的潜在高尔基定位的独特的 糖基转移酶，α 2，6-唾液酸转移酶（ST），其具有双重糖基转移酶活性。 在高尔基体和反高尔基体的反池中的定位 网络 初步证据表明，ST拥有高尔基体 在信号锚结构域和茎区中的定位信号 并且这些区域也介导酶的寡聚化。 的 这项研究计划的重点将是1）确定哪些序列在 信号锚结构域和茎区对于 ST和其他高尔基体蛋白定位于高尔基体， 2)研究寡聚化在转运和分选中的作用， 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完成， 突变和嵌合ST蛋白免疫电镜定位 通过体外诱变和PCR构建 技术. 这些研究的结果将具有根本的重要性 用于了解分泌中的蛋白质转运和分选 途径，更具体地说，对于理解高尔基体蛋白 高尔基体的定位和组装。 另外这些 研究将开始，以评估定位的分子基础， 末端糖基转移酶在高尔基体堆栈和如何这严格 糖基转移酶的区室化最终控制了结构 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.