N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.

中文翻译：

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酿酒酵母alg12delta突变体揭示了Glc3Man9GlcNAc2-PP-Dol的中臂alpha1,2Man和上臂alpha1,2Manalpha1,6Man-残基在调节内质网和高尔基体中糖蛋白聚糖加工中的作用。

通过将前体Glc（3）Man（9）GlcNAc（2）从二磷酸焦磷酸酯（PP-Dol）转移到新生蛋白质中的Asn残基，几乎所有真核生物中的N-糖基化都在内质网（ER）中进行。酿酒酵母alg（天冬酰胺连接的糖基化）突变体无法正确合成寡糖脂质，并且alg12突变体积累了Man（7）GlcNAc（2）-PP-Dol中间体。我们显示从alg12Delta分泌的转化酶释放的Man（7）GlcNAc（2）是Manalpha1,2Manalpha1,2Manalpha1,3（Manalpha1,2Manalpha1,3Manalpha1,6）-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha / beta，这证实了Man （7）GlcNAc（2）是由ALG9基因编码的中臂末端α1,2-甘露糖基转移酶的产物。尽管在alg12Delta和野生型细胞中ER葡萄糖的添加和修剪事件相似，通常由Mns1p在ER中去除的中央臂与α1，2连接的Man残基持续存在于alg12Delta背景中。这证实了体内较早的体外实验，表明在alg12Delta Man（7）GlcNAc（2）中缺失的上臂Manalpha1,2，Manalpha1,6-二糖部分被Mns1p识别并需要用于最佳甘露糖苷酶活性。该Man的存在通过降低Ochlp的效率来影响下游聚糖的加工，Ochlp是负责启动外链甘露聚糖合成的顺式高尔基体α1,6-甘露糖基转移酶，导致外部转化酶和液泡蛋白酶A的低糖基化。Mlg1p识别并需要alg12Delta Man（7）GlcNAc（2）中缺少的核苷酸来获得最佳甘露糖苷酶活性。该Man的存在通过降低Ochlp的效率来影响下游聚糖的加工，Ochlp是负责启动外链甘露聚糖合成的顺式高尔基体α1,6-甘露糖基转移酶，导致外部转化酶和液泡蛋白酶A的低糖基化。Mlg1p识别并需要alg12Delta Man（7）GlcNAc（2）中缺少的核苷酸来获得最佳甘露糖苷酶活性。该Man的存在通过降低Ochlp的效率来影响下游聚糖的加工，Ochlp是负责启动外链甘露聚糖合成的顺式高尔基体α1,6-甘露糖基转移酶，导致外部转化酶和液泡蛋白酶A的低糖基化。