ASSEMBLY AND TRANSFER OF N LINKED OLIGOSACCHARIDE

该项目的长期目标是提供对 天冬酰胺连接的结构和功能组织 粗面内质网的糖基化装置。特例 重点将放在(I)生物化学和分子 哺乳动物和真菌寡糖转移酶的特性， (Ii)酿酒酵母的鉴定和特性 功能上与基因相互作用的基因产品 寡糖转移酶和(Iii)低聚糖转移酶作用机制的研究 脂联寡糖的跨双层转运。 哺乳动物和酵母菌低聚糖转移酶的分离 微粒体膜将使用以下组合来表征 生物化学、分子和细胞生物学方法。氨基酸 酵母寡糖转移酶的亚基序列如下 由基因组克隆的分离和测序确定。这个 将对酵母寡糖转移酶亚基的功能进行评估 通过对活体条件突变的分析。空间和时间 蛋白质转运机制与蛋白质的关系 糖基化装置将在酿酒酵母中进行评估。酵母基因 与寡糖转移酶相互作用的产物将是 由基因筛查确定的高拷贝抑制子 寡糖转移酶突变体。生物合成中间体 脂联寡糖的组装已被证明是 胞浆和管腔面之间的不对称分布 粗面内质网膜。最大的中间体 在哺乳动物微粒体膜的细胞质表面检测到 (Man5GlcNAc2-PP-DOLICHOL)通过膜转运 随后延伸至Glc3Man9GlcNA2-PP-Dol。膜的拓扑结构和 脂联寡糖的跨双层转运将是 利用酿酒酵母的微粒体膜进行了研究。易位 Man5GlcNAc2-PP-Dolicol的体外研究 和从头合成的脂联寡糖作为寡糖 作为寡糖受体的供体和合成三肽。 干扰脂质相关的实验扰动 寡糖运输将被用作工具来研究 运输过程。这些研究的目的是确定 脂联低聚糖的转运是一种蛋白质介导的或 自发过程，碳水化合物缺乏性糖蛋白综合征 是一种最近被描述的多系统人类疾病，似乎 由新合成的蛋白质的多效性低糖基化引起 内质网。导致CDG的病变尚未被 定义的，但可能涉及脂质连接的组装的缺陷 寡糖供体或寡糖转移酶缺陷。因此， 这项建议中描述的研究与人类 疾病。

The long-term objective of this project is to provide insight into the structural and functional organization of the asparagine-linked glycosylation apparatus of the rough endoplasmic reticulum. Particular emphasis will be placed on (i) a biochemical and molecular characterization of the mammalian and fungal oligosaccharyltransferases, (ii) the identification and characterization of Saccharomyces cerevisiae gene products that functionally interact with the oligosaccharyltransferase and (iii) an examination of the mechanism of transbilayer transport of lipid-linked oligosaccharides. Oligosaccharyltransferase isolated from both mammalian and yeast microsomal membranes will be characterized using a combination of biochemical, molecular and cellular biological approaches. The amino acid sequence of the subunits of the yeast oligosaccharyltransferase will be determined by the isolation and sequencing of genomic clones. The function of the yeast oligosaccharyltransferase subunits will be evaluated in vivo by the analysis of conditional mutants. The spatial and temporal relationships between the protein translocation machinery and the protein glycosylation apparatus will be evaluated in S. cerevisiae. Yeast gene products that interact with the oligosaccharyltransferase will be identified by genetic screens for high-copy suppressors of an oligosaccharyltransferase mutant. Biosynthetic intermediates in the assembly of lipid-linked oligosaccharide have been shown to be asymmetrically distributed between the cytoplasmic and lumenal faces of the rough endoplasmic reticulum membrane. The largest intermediate detected upon the cytoplasmic face of mammalian microsomal membranes (Man5GlcNAc2-PP-dolichol) is transported across the membrane for subsequent elongation to Glc3Man9GlcNA2-PP-Dol. The membrane topology and transbilayer transport of lipid-linked oligosaccharides will be investigated using microsomal membranes from S. cerevisiae. Translocation of Man5GlcNAc2-PP-dolichol will be investigated in vitro using endogenous and de novo synthesized lipid-linked oligosaccharides as oligosaccharide donors and synthetic tripeptides as oligosaccharide acceptors. Experimental perturbations that interfere with lipid-linked oligosaccharide transport will be used as tools to investigate the transport process. The objective of these studies is to determine whether transport of lipid-linked oligosaccharides is a protein-mediated or spontaneous process., Carbohydrate deficient glycoprotein syndrome (CDGS) is a recently described multi-systemic human disease that appears to be caused by pleiotropic underglycosylation of newly synthesized proteins in the endoplasmic reticulum. The lesion responsible for CDGS has not been defined, but could involve a defect in the assembly of the lipid-linked oligosaccharide donor or a defect in the oligosaccharyltransferase. Thus, the research described in this proposal is of direct relevance to a human disease.