GLYCOSYLATION MECHANISMS

这项研究建议的长期目标如下： 了解调节糖基化的细胞机制 粗面内质网和高尔基体中的蛋白质和脂质 装置和（B）确定蛋白质和脂质的作用 糖基化的细胞器地形和区室化过程中 分泌和膜生物发生。 我们建议采取以下措施 实现这些目标的具体目标： (1)为了进一步研究糖基化的机制， 哺乳动物细胞的高尔基体。 我们将使用生物化学和 分子生物学方法纯化高尔基体CMPNeuAc， UDP-半乳糖转运蛋白，并克隆相应的基因。 使用我们 最近描述的蛋白脂质体重建系统，我们将使用 亲和层析和常规层析来纯化转运蛋白。 我们 也将拯救质粒，含有高尔基体UDP-半乳糖的cDNA 和CMPNeuAc转运蛋白，来自突变型中国仓鼠的转染子 缺乏上述运输活动的卵巢细胞。 针对转运蛋白的抗体将由蛋白质或 并用于免疫电镜研究， 确定转运蛋白是否在高尔基体内极化 设备. 与蛋白酶结合，抗体也将被 用于研究高尔基体中转运蛋白的排列 膜的 cDNA将用于研究 转运蛋白基因以及这些蛋白质的表达是如何调节的。 (2)继续我们对亚细胞组织的研究， 酵母高尔基体中糖基化的拓扑结构。 我们将 用由野生型DNA制成的基因组文库转化 K. lacis。最近被我们表征为缺乏UDP-GlcNAc 运输到高尔基体样囊泡，以分离和表征 高尔基体UDP-GlcNAc转运蛋白基因。 我们将克隆并破坏 高尔基体膜GDPR基因，一种来自S. 最近在我们的实验室中纯化了酿酒酵母，并假设 高尔基体甘露糖基化所必需的。 抗GDPR抗体和 酸克雷乳酸UDP-GlcNAc转运蛋白（通过其DNA序列获得） 将用于研究，（a）通过免疫电镜，它们的位置 （B）通过对蛋白酶的敏感性， 蛋白质在高尔基体膜上的分布 (3)为了继续我们对糖基化拓扑学的研究， 粗面内质网 使用膜不可渗透的探针，我们 将试图直接证明， 从细胞质侧的长轴醇-寡糖衍生物 膜进入管腔。 内质网复溶研究 内质网（ER）膜蛋白和脂质体将试图 证明了一种长链寡糖转运蛋白的存在 ER膜中的蛋白质。

The long-term goals of this research proposal are the following: (a) To understand the cellular mechanisms which regulate the glycosylation of proteins and lipids in the rough endoplasmic reticulum and Golgi apparatus and, (b) to establish the role of protein and lipid glycosylation in organelle topography and compartmentation during secretion and membrane biogenesis. We propose to pursue the following specific aims towards achieving these goals: (1) To continue with our studies on the mechanisms of glycosylation in the Golgi apparatus of mammalian cells. We will use biochemical and molecular biological approaches to purify the Golgi CMPNeuAc and UDP-Galactose transporters and to clone the respective genes. Using our recently described proteoliposome reconstitution system, we will use affinity and conventional chromatography to purify the transporters. We will also rescue plasmids, containing cDNAs of the Golgi UDP-Galactose and CMPNeuAc transporters, from transfectants of mutant Chinese hamster ovary cells, which are deficient in the above transport activities. Antibodies against the transporters will be made from the proteins or based on the cDNAs and used in immunoelectronmicroscopy studies to determine whether the transporters are polarized within the Golgi apparatus. In conjunction with proteases, the antibodies will also be used to study the arrangement of the transporter proteins in the Golgi membrane. The cDNAs will be used to study the structure of the transporter genes and how the expression of these proteins is regulated. (2) To continue with our studies on the subcellular organization and topography of glycosylation in the Golgi apparatus of yeast. We will transform, with a genomic library made from wild-type DNA, a mutant of K. lacis. recently characterized by us to be deficient in UDP-GlcNAc transport into Golgi-like vesicles in order to isolate and characterize the Golgi UDP-GlcNAc transporter gene. We will clone and disrupt the gene of the Golgi membrane GDPase, a lumenal marker enzyme from S. cerevisiae recently purified in our laboratory and hypothesized to be necessary for Golgi mannosylation. Antibodies against the GDPase and the K. lactis UDP-GlcNAc transporters (obtained via their DNA sequence) will be used to study, (a) by immunoelectronmicroscopy, their location within the cell, and (b) via sensitivity towards proteases to establish the proteins' topography in the Golgi membrane. (3) To continue with our studies on the topography of glycosylation in the rough endoplasmic reticulum. Using membrane impermeable probes, we will attempt to demonstrate, directly, translocation of dolichol-oligosaccharide derivatives from the cytosolic side of the membrane into the lumen. Reconstitution studies with endoplasmic reticulum (ER) membrane proteins and liposomes will be attempted to demonstrate the occurrence of a dolichol-oligosaccharide translocator protein in the ER membme.