Chavan Manasi, Chen Zhiqiang, Li Guangtao, Schindelin Hermann, Lennarz William J, Li Huilin
Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, NY 11794, USA.
Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):8947-52. doi: 10.1073/pnas.0603262103. Epub 2006 Jun 5.
The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of approximately 500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-A resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation.
寡糖基转移酶（OT）复合物在内质网腔中催化N-糖基化。酵母OT复合物由九个亚基组成，所有亚基均为跨膜蛋白。包括我们之前的分裂泛素研究在内的多项证据表明OT复合物具有寡聚结构，但确切的寡聚性质尚不清楚。通过对OT复合物的Ost4p亚基进行FLAG表位标记，我们使用非变性去污剂洋地黄皂苷和一步免疫亲和技术纯化了OT酶复合物。洋地黄皂苷溶解的OT复合物具有催化活性，并且酶复合物中存在所有九个亚基。纯化的OT复合物的表观质量约为500 kDa，表明为二聚体构型，这一点通过生化研究得到了证实。电子显微镜显示出均匀的单个颗粒，并揭示了OT复合物的二聚体结构，这与我们的生化研究一致。从电子显微镜图像重建了分辨率为25埃的二聚体OT复合物的三维结构。我们认为，OT的二聚体结构可能是与转位体二聚体有效结合以及在共翻译糖基化过程中进行变构调节所必需的。
