CYTOPLASMIC FUCOSYLATION

蛋白质糖基化的突变对细胞生长有显著影响， 增殖率，细胞分选，假原质团迁移，孢子衣 形成，孢子萌发和蛋白质运输速率，在细胞内 盘基网柄黏菌 该项目的长期目标是 在分子水平上理解糖基化是如何支持这些 正常细胞中的细胞活动。 分析一种突变体， GDP-岩藻糖合成的条件已经证明了最佳细胞 增殖速率和孢子健康取决于正常的岩藻糖基化。 对可能影响这些过程的岩藻糖基化途径的检查 导致发生岩藻糖基化反应的部分特征， 在网骨藻细胞质中。 因为他们的 区室化，受体岩藻蛋白（FP 21），和 修饰它的岩藻糖基转移酶（FP 21-岩藻糖基转移酶）， 特别准备好影响这些行为。 生化研究 分离的细胞导致了FP 21在细胞中积累的结论。 细胞质中，活性FP 21-岩藻糖基转移酶在细胞质中积累， 岩藻糖基转移酶对细胞质具有非常高的亲和力， 糖核苷酸供体，GDP-岩藻糖。 这导致了一种 模型表明FP 21在细胞质中合成并积累，并且 它被合成的驻留糖基转移酶糖基化， 在那里，从糖核苷酸的细胞质池。 具体 待检验的假设是FP 21在细胞质中合成， 它在细胞质中被α（1-4）岩藻糖基转移酶岩藻糖基化， 也是在那里合成的，FP 21具有独特的结构特征 将其标记为FTase的岩藻糖基受体，并且该反应 在其他生物体中保存。 这些假设将由以下人员进行检验： 开发抗体以研究FP 21的生物发生区室， 岩藻糖基转移酶，利用免疫显微镜确认定位 在完整的细胞中，比较预测的 初级翻译产物与已知的细胞质、非膜 蛋白质，确定FP 21寡糖的结构，以及 对岩藻糖基转移酶反应进行竞争研究。 一旦 FP 21及其岩藻糖基转移酶以这些方式定义， 直接测试它们在增殖和孢子健康中的作用， 识别在其他生物中以这种方式修饰的糖蛋白。 了解细胞质糖基化是很重要的，因为它 巨大的监管潜力，这一点可以从以下方面的作用中得到证明： Ser和Thr残基的磷酸化对蛋白质的活性和构象的影响 改性蛋白质 描述这一途径的第二个原因涉及 由于对岩藻缀合物的分析日益关注， 活组织检查和生物液体中的岩藻糖基化活性，作为 病理学中的细胞活动。 对这些问题的理性解释 分析将受益于对多重潜力的理解， 这些物质的细胞来源。

Mutations in the glycosylation of proteins have dramatic effects on cell proliferation rate, cell sorting, pseudoplasmodial migration, spore coat formation, spore germination, and protein transport rate, in the cellular slime mold Dictyostelium discoideum. The long term goal of the project is to understand at the molecular level how glycosylation supports these cellular activities in normal cells. Analysis of a mutant that is conditional for GDP-Fucose synthesis has demonstrated that optimal cell proliferation rate and spore health depend on normal fucosylation. Examination of fucosylation pathways that may influence these processes has led to the partial characterization of a fucosylation reaction which occurs in the cytoplasm of Dictyostelium cells. Because of their compartmentalization, the acceptor fucoprotein (FP21), and the fucosyltransferase which modifies it (FP21-fucosyltransferase), are specially poised to influence these behaviors. Biochemical studies on fractionated cells have led to the conclusion that FP21 accumulates in the cytoplasm, that active FP21-fucosyltransferase accumulates in the cytoplasm, and that the fucosyltransferase has a very high affinity for the sugar nucleotide donor, GDP-Fucose. This has led to the development of a model that FP21 is synthesized and accumulates in the cytoplasm, and that it is glycosylated by resident glycosyltransferases, which are synthesized there, from cytoplasmic pools of sugar nucleotides. The specific hypotheses to be tested are that FP21 is synthesized in the cytoplasm, that it is fucosylated in the cytoplasm by an alpha(1-4)fucosyltransferase which is also synthesized there, that FP21 possesses unique structural features that mark it as a fucosyl acceptor for the FTase, and that this reaction is conserved in other organisms. These hypotheses will be tested by developing antibodies to study the compartments of biogenesis of FP21 and the fucosyltransferase, utilizing immunomicroscopy to confirm localization in intact cells, comparing the predicted amino acid sequences of the primary translation products with those of known cytoplasmic, non-membrane proteins, determining the structure of the FP21 oligosaccharide, and carrying out competition studies on the fucosyltransferase reaction. Once FP21 and its fucosyltransferase are defined in these ways, there will be a basis to directly test their role in proliferation and spore health, and to identify glycoproteins that are modified in this way in other organisms. It is important to understand cytoplasmic glycosylation because of its tremendous regulatory potential, as evidenced by the roles of phosphorylation of Ser and Thr residues for activity and conformation of modified proteins. A second reason for characterizing this pathway relates to the increasing attention given to the analysis of fucoconjugates and fucosylation activities in biopsies and biological fluids, as markers for cellular activity in pathology. A rational interpretation of these analyses would benefit from an understanding of the multiple potential cellular origins of these substances.