Interactions among subunits of the oligosaccharyltransferase complex
Interactions among subunits of the oligosaccharyltransferase complex
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DOI：
10.1074/jbc.272.47.29687
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发表时间：
1997-11-21
影响因子：
4.8
通讯作者：
Kreibich, G
中科院分区：
文献类型：
--
作者：
Fu, J;Ren, MD;Kreibich, G
哺乳动物低聚糖转移酶（OST）是一种由三种内质网膜蛋白组成的低聚复合物：核糖素I （RI）、核糖素II （RII）和OST48。此外，酵母OST复合体的Ost2亚基与Dad1（抗凋亡死亡的防御者）之间的序列同源性表明Dad1可能是哺乳动物OST复合体的第四个亚基。为了阐明这种复合体的结构组织，我们研究了其亚基之间的相互作用。利用酵母双杂交系统，我们发现RI和RII的管腔结构域（分别为Ra和RIIL）与OST48的管腔结构域（OST48L）相互作用，但RIL和RIIL之间没有直接相互作用。这些结果经生化试验证实。利用酵母双杂交系统进行的缺失分析表明，相邻跨膜结构域的RIL或RIIL子结构域与OST48L相互作用。在OST48L的三个等长亚结构域中，位于N端和靠近跨膜结构域的一个与RIL相互作用。OST48L的这三个子域都不与RIIL相互作用。酵母双杂交实验还发现Dad1的细胞质n端区域与OST48的细胞质短尾之间存在亲和力，从而将Dad1牢牢地置于OST复合体中。此外，我们还发现了RI细胞质结构域之间的同型相互作用，这可能在由易位机制组成的低聚物阵列的形成中发挥作用。
The mammalian oIigosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (Ra and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. Tn addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.