GLYCOSYLATION MECHANISMS

这项研究建议的长期目标如下：(A) 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 以及，(B)确定蛋白质和脂肪的作用 细胞器形态和区划过程中的糖基化 分泌物与膜生物发生。我们建议进行以下工作 实现这些目标的具体目标： (1)继续研究糖基化的机制。 哺乳动物细胞的高尔基体。我们将使用生化和 高尔基体CMPNeuAc和CMPNeuAc的分子生物学纯化方法 UDP-半乳糖转运体和克隆各自的基因。使用我们的 最近描述的蛋白质脂质体重建系统，我们将使用 亲和层析和常规层析纯化转运蛋白。我们 也将拯救含有高尔基UDP-半乳糖cDNA的质粒 和CMPNeuAc转运蛋白 缺乏上述转运活动的卵巢细胞。 针对转运蛋白的抗体将由蛋白质或 基于cDNA并用于免疫电子显微镜研究 确定高尔基体内的转运体是否极化 仪器。结合蛋白水解酶，抗体也将被 用于研究高尔基体转运蛋白的排列 薄膜。这些DNA将被用来研究 转运蛋白基因以及这些蛋白的表达是如何被调控的。 (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图。我们会 利用野生型DNA组成的基因组文库进行转化，获得了一个突变体 K.lacis。最近被我们描述为缺乏UDP-GlcNAc 转运入类高尔基体囊泡以分离和鉴定 高尔基体UDP-GlcNAc转运蛋白基因。我们将克隆并颠覆 高尔基膜GDPase基因是S。 最近在我们实验室提纯的酿酒酵母，推测是 是高尔基人甘露糖化所必需的。抗GDPase和GDPase抗体 乳酸杆菌UDP-GlcNAc转运蛋白(通过DNA测序获得) 将被用来研究(A)通过免疫电子显微镜，它们的位置 在细胞内，以及(B)通过对蛋白酶的敏感性来建立 高尔基体膜上蛋白质的形态。 (3)继续我们对糖基化构象的研究 粗面内质网。使用不透膜的探头，我们 将试图直接证明，移位 二羟基苯二醇寡糖衍生物来自胞液侧 膜进入管腔。内质重建研究 将尝试网状(ER)膜蛋白和脂质体 证明出现了二醇低聚糖转运体 内质网中的蛋白质。

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.