Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain sec61, TRAP, and two potential new subunits
Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain sec61, TRAP, and two potential new subunits
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DOI：
10.1021/bi047328f
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发表时间：
2005-04-26
期刊：
影响因子：
2.9
通讯作者：
Skach, WR
中科院分区：
文献类型：
--
作者：
Shibatani, T;David, LL;Skach, WR
寡糖基转移酶（OST）催化粗面内质网腔内N-连接糖基化过程中高甘露糖共翻译转移为新生多肽。已经在酵母中鉴定了九个OST亚基。然而，哺乳动物OST的组成和组织仍不清楚。使用二维蓝色天然聚丙烯酰胺凝胶电泳/十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和质谱，我们现在证明，哺乳动物OST可以从溶解的，积极从事核糖体作为多个不同的蛋白质复合物，大小范围从500至700 kDa的类似分离。这些复合物表现出不同的核糖体亲和力和亚基组成。主要的复合物，OSTC 1，具有类似于500 kDa的表观大小，并且在生理盐条件下嘌呤霉素处理后容易从核糖体转位子复合物中释放。仅在用高盐处理后释放两种另外的复合物：OSTC II（类似于600 kDa）和OSTC III（类似于700 kDa）。两者都保持与异源三聚体Sec 61 α β γ稳定结合，而OSTC III也含有四聚体TRAP复合物。所有已知的哺乳动物OST亚基（STT 3-A、ribophorin I、ribophorin II、OST 48和DAD 1）都存在于所有复合物中。此外，还用OST共纯化了两种以前未表征的蛋白质。质谱鉴定了一个17 kDa的蛋白为DC 2，它与酵母Ost 3 p和Ost 6p的C-末端半部分弱同源。第二种蛋白质（14 kDa）被初步鉴定为角质形成细胞相关蛋白2（KCP 2），并且没有先前已知的功能。我们的研究结果确定了两个潜在的新的哺乳动物OST亚基，并证明了显着的异质性OST组合物，可能反映了控制新生链糖基化的手段。
Oligosaccharyltransferase (OST) catalyzes the cotranslational transfer of high-mannose sugars to nascent polypeptides during N-linked glycosylation in the rough endoplasmic reticulum lumen. Nine OST subunits have been identified in yeast. However, the composition and organization of mammalian OST remain unclear. Using two-dimensional Blue Native polyacrylamide gel electrophoresis/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, we now demonstrate that mammalian OST can be isolated from solubilized, actively engaged ribosomes as multiple distinct protein complexes that range in size from similar to 500 to 700 kDa. These complexes exhibit different ribosome affinities and subunit compositions. The major complex, OSTC1, had an apparent size of similar to 500 kDa and was readily released from ribosome translocon complexes after puromycin treatment under physiological salt conditions. Two additional complexes were released only after treatment with high salt: OSTCII (similar to 600 kDa) and OSTCIII (similar to 700 kDa). Both remained stably associated with heterotrimeric Sec61 alpha beta gamma, while OSTCIII also contained the tetrameric TRAP complex. All known mammalian OST subunits (STT3-A, ribophorin I, ribophorin II, OST48, and DAD1) were present in all complexes. In addition, two previously uncharacterized proteins were also copurified with OST. Mass spectrometry identified a 17 kDa protein as DC2 which is weakly homologous to the C-terminal half of yeast Ost3p and Ost6p. The second protein (14 kDa) was tentatively identified as keratinocyte-associated protein 2 (KCP2) and has no previously known function. Our results identify two potential new subunits of mammalian OST and demonstrate a remarkable heterogeneity in OST composition that may reflect a means for controlling nascent chain glycosylation.