Sanjay A, Fu J, Kreibich G
Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
J Biol Chem. 1998 Oct 2;273(40):26094-9. doi: 10.1074/jbc.273.40.26094.
Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex.
天冬酰胺连接的糖基化是一种在所有真核生物中都发生的高度保守的蛋白质修饰反应。寡糖基转移酶（OST）的活性位点暴露在内质网（ER）的腔面上，它催化预组装的高甘露糖寡糖转移到新生多肽的某些天冬酰胺残基上。哺乳动物的OST复合物最初被认为由三种跨膜蛋白组成，即核糖体结合蛋白I（RI）、核糖体结合蛋白II（RII）和OST48。最近，一种名为DAD1的12 kDa小整合膜蛋白被鉴定为哺乳动物OST复合物的另一个成员。DAD1基因中的一个点突变导致了一种源自幼仓鼠肾的细胞系（tsBN7）的温度敏感表型，该细胞系在非允许温度下会发生凋亡。此外，在将细胞转移到非允许温度6小时后，tsBN7细胞中检测不到突变蛋白DAD1。这种温度敏感细胞系为研究一个OST亚基的缺失对其他三个亚基以及对N-连接糖基化的影响提供了独特的机会。对细胞裂解物的蛋白质印迹分析表明，在非允许温度下6小时后，核糖体结合蛋白的稳态水平降低了约50%，而OST48几乎检测不到。另一方面，粗面内质网的其他成分，如转位相关膜蛋白（TRAP）的α亚基和作为转位装置成分的Sec61复合物，其稳态水平不受OST亚基不稳定性的影响。此外，在将细胞转移到非允许温度6小时后，核糖体结合蛋白的N-糖基化受到严重影响，12小时后它们仅以非糖基化形式合成。正如所预期的那样，在非允许温度下OST复合物的这种缺陷也导致了一种分泌性糖蛋白的糖基化不足。我们得出结论，在非允许温度下DAD1的降解不仅影响OST48和核糖体结合蛋白的稳定性，还导致OST复合物的功能失活。
