Huffaker T C, Robbins P W
J Biol Chem. 1982 Mar 25;257(6):3203-10.
A [3H]mannose suicide selection has been used to isolate mutants in yeast which contain temperature-sensitive defects in asparagine-linked glycosylation. The surviving cells were screened at the nonpermissive temperature for a decreased ability to incorporate [3H]mannose and for defects in glycosylation of the secreted protein invertase. One of these mutants (alg1-1) has been characterized and found to be blocked in the assembly of the lipid-linked oligosaccharide precursor. The alg1-1 cells synthesize mannosyl compounds at 60% of the wild type level at the nonpermissive temperature and 105% of the wild type level at the permissive temperature. In vivo labeling experiments have demonstrated that alg1-1 cells are able to synthesize GlcNAc2-lipid but are unable to synthesize any mannose-containing oligosaccharide-lipids. This result was confirmed by in vitro labeling of yeast membranes. When incubated with UDP-[3H]GlcNAc, alg1-1 membranes synthesized GlcNAc2-lipid but failed to elongate it when GDP-Man was added. The alg1-1 membranes also failed to elongate exogenous GlcNAc2-lipid but were able to convert Man1GlcNAc2-lipid to Man5-Glc-NAc2-lipid in the presence of GDP-Man. These results indicate that the alg1-1 mutant is blocked specifically in the addition of the first mannose residue to the lipid-linked oligosaccharide precursor.
利用[3H]甘露糖自杀筛选法分离酵母中的突变体，这些突变体在天冬酰胺连接的糖基化过程中存在温度敏感缺陷。在非允许温度下筛选存活细胞，以检测其掺入[3H]甘露糖能力的降低以及分泌蛋白转化酶糖基化的缺陷。其中一个突变体（alg1-1）已被鉴定，发现其在脂质连接的寡糖前体组装过程中受阻。alg1-1细胞在非允许温度下合成的甘露糖基化合物水平为野生型的60%，在允许温度下为野生型的105%。体内标记实验表明，alg1-1细胞能够合成GlcNAc2-脂质，但无法合成任何含甘露糖的寡糖-脂质。酵母膜的体外标记实验证实了这一结果。当与UDP-[3H]GlcNAc一起孵育时，alg1-1膜合成了GlcNAc2-脂质，但添加GDP-Man后无法使其延长。alg1-1膜也无法延长外源性GlcNAc2-脂质，但在GDP-Man存在的情况下能够将Man1GlcNAc2-脂质转化为Man5-Glc-NAc2-脂质。这些结果表明，alg1-1突变体在脂质连接的寡糖前体上添加第一个甘露糖残基的过程中被特异性阻断。
