DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase
DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase
复制标题
DOI：
10.1073/pnas.94.10.4994
复制
发表时间：
1997-05-13
影响因子：
11.1
通讯作者：
Gilmore, R
中科院分区：
文献类型：
--
作者：
Kelleher, DJ;Gilmore, R
DAD1是抗凋亡细胞死亡的防御者，最初在bhk21衍生的tsBN7细胞系中被确定为程序性细胞死亡的负调节因子，有趣的是，12.5 kda的DAD1蛋白与酵母寡糖转移酶（OST）的16 kda亚基Ost2p在序列上有40%相同，尽管后者的观察表明DAD1可能是哺乳动物OST亚基，但支持这一假设的生化证据尚未报道。我们发现犬的OST活性与核糖素I、核糖素II和OST48的寡聚复合物有关。在这里，我们证明DAD1在完整的膜和纯化的酶中都是与OST紧密相关的亚基，对洗涤剂溶解的WI38细胞和犬粗粒体的沉降速度分析表明，DAD1与OST活性、核糖素和OST48精确地共同沉积，纯化的OST的放射性碘化显示DAD1的存在量与其他亚基大致相当。DAD1可以在完整微粒体中与二硫代二（琥珀酰丙酸）交联至OST48。DAD1-核糖素II-OST48异二聚体、DAD1-核糖素II-OST48异三聚体和DAD1-核糖素i -核糖素II-OST48异四聚体也被检测到。DAD1是OST的一个亚基的证明表明，在tsBN7细胞系中，DAD1缺失诱导细胞死亡途径反映了真核生物n -链糖基化的本质。
DAD1, the defender against apoptotic cell death, was initially identified as a negative regulator of programmed cell death in the BHK21-derived tsBN7 cell line, Of interest, the 12.5-kDa DAD1 protein is 40% identical in sequence to Ost2p, the 16-kDa subunit of the yeast oligosaccharyltransferase (OST), Although the latter observation suggests that DAD1 may be a mammalian OST subunit, biochemical evidence to support this hypothesis has not been reported, Previously, we showed that canine OST activity is associated with an oligomeric complex of ribophorin I, ribophorin II, and OST48. Here, we demonstrate that DAD1 is a tightly associated subunit of the OST both in the intact membrane and in the purified enzyme, Sedimentation velocity analyses of detergent-solubilized WI38 cells and canine rough microsomes show that DAD1 cosediments precisely with OST activity and with the ribophorins and OST48, Radioiodination of the purified OST reveals that DAD1 is present in roughly equimolar amounts relative to the other subunits, DAD1 can be crosslinked to OST48 in intact microsomes with dithio-bis(succinimidylpropionate) . Crosslinked ribophorin II-OST48 heterodimers, DAD1-ribophorin II-OST48 heterotrimers and DAD1-ribophorin I-ribophorin II-OST48 heterotetramers also were detected, The demonstration that DAD1 is a subunit of the OST suggests that induction of a cell death pathway upon loss of DAD1 in the tsBN7 cell line reflects the essential nature of N-linked glycosylation in eukaryotes.