Runge K W, Huffaker T C, Robbins P W
J Biol Chem. 1984 Jan 10;259(1):412-7.
Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.
通过[3H]甘露糖自杀富集法，已分离出脂质连接寡糖生物合成中的两个互补突变体。突变体alg5-1和alg6-1在体内和体外积累的最大脂质连接寡糖不是野生型前体寡糖Glc3man9GlcNA2-P-P-多萜醇，而是Man9GlcNAc2-P-P-多萜醇。当向每个突变体的微粒体膜中添加UDP-[3H]Glc时，两者都不能延长Man9GlcNAc2-P-P-多萜醇，并且只有alg6-1能够合成多萜醇磷酸葡萄糖。当将多萜醇磷酸[3H]葡萄糖添加到alg5-1、alg6-1或亲本菌株的微粒体中时，只有alg5-1和亲本菌株能合成糖基化的脂质连接寡糖。这些结果表明，alg5-1细胞无法合成多萜醇磷酸葡萄糖，而alg6-1细胞无法将葡萄糖从多萜醇磷酸葡萄糖转移到未糖基化的脂质连接寡糖上。我们还提供了证据表明这两个突变体都能将Man9GlcNAc2转移到蛋白质上。
