Roos J, Sternglanz R, Lennarz W J
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215.
Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1485-9. doi: 10.1073/pnas.91.4.1485.
Dolichol in the form of dolichyl phosphate participates in the synthesis of N- and O-linked glycoproteins and phosphatidylinositol-linked proteins in the yeast Saccharomyces cerevisiae. In this organism, as well as in higher eukaryotes, a number of the enzymes in the polyisoprenoid and glycoprotein biosynthetic pathways have not been identified. In this study, we have developed a convenient, highly sensitive assay that uses one of the end products of the dolichylphosphate synthetic pathway, oligosaccharide-diphosphodolichol, and a 125I-labeled peptide substrate for N-linked glycosylation to screen a collection of temperature-sensitive yeast mutants for defects in protein glycosylation. By using a combination of biochemical and genetic procedures, the defective mutants were grouped into three categories: those containing defects in dolichyl-phosphate synthesis (class 1), lipid-linked oligosaccharide assembly (class 2), or oligosaccharide transferase activity (class 3). Among the mutants identified by this screen were sec59 (which encodes dolichol kinase) and a mutant that affects the activity of the ALG1-encoded mannosyltransferase that forms dolichol-PP-(GlcNAc)2Man1. Of particular interest was a mutant that exhibits a temperature-sensitive defect in oligosaccharide transferase activity. This mutant, meg1 (microsomal protein essential for glycosylation 1) assembles a complete oligosaccharide chain and, therefore, is likely to be a class 3 mutant. We report the cloning of MEG1, the gene that rescues the oligosaccharide transferase activity defect in this mutant. A number of criteria distinguish this gene from previously described genes in this pathway.
磷酸多萜醇形式的多萜醇参与酿酒酵母中N-连接和O-连接糖蛋白以及磷脂酰肌醇连接蛋白的合成。在这种生物体以及高等真核生物中，多聚异戊二烯和糖蛋白生物合成途径中的许多酶尚未被鉴定出来。在本研究中，我们开发了一种简便、高灵敏度的检测方法，该方法使用磷酸多萜醇合成途径的终产物之一——寡糖二磷酸多萜醇，以及用于N-连接糖基化的125I标记肽底物，来筛选温度敏感型酵母突变体文库，以寻找蛋白质糖基化缺陷。通过结合生化和遗传学方法，将有缺陷的突变体分为三类：那些在磷酸多萜醇合成（第1类）、脂连接寡糖组装（第2类）或寡糖转移酶活性（第3类）方面存在缺陷的突变体。通过该筛选鉴定出的突变体包括sec59（编码多萜醇激酶）和一个影响由ALG1编码的形成多萜醇-PP-(GlcNAc)2Man1的甘露糖基转移酶活性的突变体。特别令人感兴趣的是一个在寡糖转移酶活性方面表现出温度敏感缺陷的突变体。这个突变体meg1（糖基化必需的微粒体蛋白1）组装了完整的寡糖链，因此，它可能是一个第3类突变体。我们报告了MEG1基因的克隆，该基因挽救了该突变体中的寡糖转移酶活性缺陷。许多标准将该基因与该途径中先前描述的基因区分开来。
